Optical system and assay chip for probing, detecting and analyzing molecules

ABSTRACT

Apparatus and methods for analyzing single molecule and performing nucleic acid sequencing. An apparatus can include an assay chip that includes multiple pixels with sample wells configured to receive a sample, which, when excited, emits emission energy; at least one element for directing the emission energy in a particular direction; and a light path along which the emission energy travels from the sample well toward a sensor. The apparatus also includes an instrument that interfaces with the assay chip. The instrument includes an excitation light source for exciting the sample in each sample well; a plurality of sensors corresponding the sample wells. Each sensor may detect emission energy from a sample in a respective sample well. The instrument includes at least one optical element that directs the emission energy from each sample well towards a respective sensor of the plurality of sensors.

RELATED APPLICATIONS

This application is a continuation and claims the benefit under 35U.S.C. § 120 of U.S. patent application Ser. No. 15/808,940, entitled“Optical System and Assay Chip for Probing, Detecting and AnalyzingMolecules” filed Nov. 10, 2017, which is a continuation and claims thebenefit under 35 U.S.C. § 120 of U.S. patent application Ser. No.14/543,867, entitled “Optical System and Assay Chip for Probing,Detecting and Analyzing Molecules” filed on Nov. 17, 2014, which claimsthe benefit under 35 U.S.C. § 119(e) of U.S. Provisional PatentApplication 61/905,282, entitled “Integrated Device for Probing,Detecting and Analyzing Molecules,” filed Nov. 17, 2013; U.S.Provisional Patent Application 61/917,926, entitled “Integrated Devicefor Probing, Detecting and Analyzing Molecules,” filed Dec. 18, 2013;and U.S. Provisional Patent Application 61/941,916, entitled “IntegratedDevice for Probing, Detecting and Analyzing Molecules,” filed Feb. 19,2014, each of which is incorporated by reference in their entirety.

BACKGROUND Field

The present application is directed generally to devices, methods andtechniques for performing rapid, massively parallel, quantitativeanalysis of biological and/or chemical samples, and methods offabricating said devices.

Related Art

Detection and analysis of biological samples may be performed usingbiological assays (“bioassays”). Bioassays conventionally involve large,expensive laboratory equipment requiring research scientists trained tooperate the equipment and perform the bioassays. Moreover, bioassays areconventionally performed in bulk such that a large amount of aparticular type of sample is necessary for detection and quantitation.

Some bioassays are performed by tagging samples with luminescent tagsthat emit light of a particular wavelength. The tags are illuminatedwith an excitation light source to cause luminescence, and theluminescent light is detected with a photodetector to quantify theamount of luminescent light emitted by the tags. Bioassays usingluminescent tags conventionally involve expensive laser light sources toilluminate samples and complicated, bulky detection optics andelectronics to collect the luminescence from the illuminated samples.

SUMMARY

The technology described herein relates to apparatus and methods foranalyzing specimens rapidly using an assay chip and instrument. Theassay chip may be in the form of a disposable or recyclable chip that isconfigured to receive a small amount of a specimen and execute, inparallel, a large number of analyses of samples within the specimen. Theassay chip and instrument may be used to detect the presence ofparticular chemical or biological analytes in some embodiments, toevaluate a chemical or biological reactions in some embodiments, and todetermine genetic sequences in some embodiments. According to someimplementations, the integrated device may be used for single-moleculegene sequencing.

According to some implementations, a user deposits a specimen in achamber on the assay chip, and inserts the assay chip into a receivinginstrument. The instrument, alone or in communication with a computer,automatically interfaces with the integrated device, sends and receiveslight from the assay chip, detects and processes the received light, andprovides results of the analysis to the user.

According to some embodiments, an assay chip includes a sample wellconfigured to receive a sample, which, when excited, emits emissionenergy; at least one element that directs the emission energy in aparticular direction; and a light path along which the emission energytravels from the sample well toward a sensor. The at least one elementis selected from the group consisting of a refractive element, adiffractive element, a plasmonic element and a resonator.

According to some embodiments, an instrument configured to interfacewith an assay chip including a plurality of sample wells, each samplewell of the plurality of sample wells configured to receive a sample,the instrument include at least one excitation light source configuredto excite the sample of at least a portion of the plurality of samplewells; a plurality of sensors, each sensor of the plurality of sensorscorresponding to a sample well of the plurality of sample wells, whereineach sensor of the plurality of sensors is configured to detect emissionenergy from the sample in a respective sample well; and at least oneoptical element configured to direct the emission energy from eachsample well of the plurality of sample wells towards a respective sensorof the plurality of sensors.

According to some embodiments, an apparatus includes an assay chipincluding a plurality of pixels and an instrument configured tointerface with the assay chip. Each of the plurality of pixels of theassay chip includes a sample well configured to receive a sample, which,when excited, emits emission energy; at least one element for directingthe emission energy in a particular direction, wherein the at least oneelement is selected from the group consisting of a refractive element, adiffractive element, a plasmonic element and a resonator; and a lightpath along which the emission energy travels from the sample well towarda sensor. The instrument includes at least one excitation light sourceconfigured to excite the sample in each sample well; a plurality ofsensors, each sensor of the plurality of sensors corresponding to arespective sample well, wherein each sensor of the plurality of sensorsis configured to detect emission energy from the sample in therespective sample well; and at least one optical element configured todirect the emission energy from each sample well towards a respectivesensor of the plurality of sensors.

According to some embodiments, a method of analyzing a specimen includesproviding the specimen on the top surface of an assay chip comprising aplurality of sample wells; aligning the chip with an instrumentcomprising at least one excitation light source and at least one sensor;exciting a sample from the specimen in at least one of the plurality ofsample wells with excitation light from the at least one excitationlight source; and detecting, with the at least one sensor, emissionenergy generated by the sample in the at least one sample well inresponse to excitation by the excitation light.

According to some embodiments, a method for sequencing a target nucleicacid molecule includes: (a) providing a chip adjacent to an instrumentthat includes an excitation source and a sensor, wherein said chipincludes at least one well that is operatively coupled to saidexcitation source and said sensor when said chip is at a sensingposition of said instrument, and wherein said well contains said targetnucleic acid molecule, a polymerizing enzyme and a plurality of types ofnucleotides or nucleotide analogs; (b) with said chip at said sensingposition, performing an extension reaction at a priming location of saidtarget nucleic acid molecule in the presence of said polymerizing enzymeto sequentially incorporate said nucleotides or nucleotide analogs intoa growing strand that is complementary to said target nucleic acidmolecule, wherein upon incorporation and excitation by excitation energyfrom said excitation source, said nucleotides or nucleotides analogsemit signals in said well; (c) using said sensor to detect spatialand/or temporal distribution patterns of said signals that aredistinguishable for said plurality of types of nucleotides or nucleotideanalogs; and (d) identifying said nucleotides or nucleotide analogsbased on said spatial and/or temporal distribution patterns of saidsignals, thereby sequencing said target nucleic acid molecule.

According to some embodiments, a method for nucleic acid sequencingincludes: (a) providing a chip adjacent to an instrument, wherein saidchip includes a plurality of wells that are each operatively coupled to(i) an excitation source and (ii) a sensor of said instrument when saidchip is at a sensing position of said instrument, and wherein anindividual well of said plurality contains said target nucleic acidmolecule, a polymerizing enzyme and a plurality of types of nucleotidesor nucleotide analogs; (b) with said chip at said sensing position,subjecting said target nucleic acid molecule to a polymerizationreaction to yield a growing strand that is complementary to said targetnucleic acid molecule in the presence of said nucleotides or nucleotideanalogs and said polymerizing enzyme, wherein said nucleotides ornucleotides analogs emit signals in said individual well upon excitationby excitation energy from said excitation source during incorporation;(c) using said sensor to detect spatial and/or temporal distributionpatterns of said signals that are distinguishable for said plurality oftypes of nucleotides or nucleotide analogs; and (d) identifying asequence of said target nucleic acid molecule based on said spatialand/or temporal distribution patterns of said signals.

The foregoing and other aspects, embodiments, and features of thepresent teachings can be more fully understood from the followingdescription in conjunction with the accompanying drawings.

The term “pixel” may be used in the present disclosure to refer to aunit cell of an integrated device. The unit cell may include a samplewell and a sensor. The unit cell may further include an excitationsource. The unit cell may further include at least oneexcitation-coupling optical structure (which may be referred to as a“first structure”) that is configured to enhance coupling of excitationenergy from the excitation source to the sample well. The unit cell mayfurther include at least one emission-coupling structure that isconfigured to enhance coupling of emission from the sample well to thesensor. The unit cell may further include integrated electronic devices(e.g., CMOS devices). There may be a plurality of pixels arranged in anarray on an integrated device.

The term “optical” may be used in the present disclosure to refer tovisible, near infrared, and short-wavelength infrared spectral bands.

The term “tag” may be used in the present disclosure to refer to a tag,probe, marker, or reporter attached to a sample to be analyzed orattached to a reactant that may be reacted with a sample.

The phrase “excitation energy” may be used in the present disclosure torefer to any form of energy (e.g., radiative or non-radiative) deliveredto a sample and/or tag within the sample well. Radiative excitationenergy may comprise optical radiation at one or more characteristicwavelengths.

The phrase “characteristic wavelength” may be used in the presentdisclosure to refer to a central or predominant wavelength within alimited bandwidth of radiation. In some cases, it may refer to a peakwavelength of a bandwidth of radiation. Examples of characteristicwavelengths of fluorophores are 563 nm, 595 nm, 662 nm, and 687 nm.

The phrase “characteristic energy” may be used in the present disclosureto refer to an energy associated with a characteristic wavelength.

The term “emission” may be used in the present disclosure to refer toemission from a tag and/or sample. This may include radiative emission(e.g., optical emission) or non-radiative energy transfer (e.g., Dexterenergy transfer or Förster resonant energy transfer). Emission resultsfrom excitation of a sample and/or tag within the sample well.

The phrase “emission from a sample well” or “emission from a sample” maybe used in the present disclosure to refer to emission from a tag and/orsample within a sample well.

The term “self-aligned” may be used in the present disclosure to referto a microfabrication process in which at least two distinct elements(e.g., a sample well and an emission-coupling structure, a sample welland an excitation-source) may be fabricated and aligned to each otherwithout using two separate lithographic patterning steps in which afirst lithographic patterning step (e.g., photolithography, ion-beamlithography, EUV lithography) prints a pattern of a first element and asecond lithographic patterning step is aligned to the first lithographicpatterning step and prints a pattern of the second element. Aself-aligned process may comprise including the pattern of both thefirst and second element in a single lithographic patterning step, ormay comprise forming the second element using features of a fabricatedstructure of the first element.

The term “sensor” may be used in the present disclosure to refer to oneor more integrated circuit devices configured to sense emission from thesample well and produce at least one electrical signal representative ofthe sensed emission.

The term “nano-scale” may be used in the present disclosure to refer toa structure having at least one dimension or minimum feature size on theorder of 150 nanometers (nm) or less, but not greater than approximately500 nm.

The term “micro-scale” may be used in the present disclosure to refer toa structure having at least one dimension or minimum feature sizebetween approximately 500 nm and approximately 100 microns.

The phrase “enhance excitation energy” may be used in the presentdisclosure to refer to increasing an intensity of excitation energy atan excitation region of a sample well. The intensity may be increased byconcentrating and/or resonating excitation energy incident on the samplewell, for example. In some cases, the intensity may be increased byanti-reflective coatings or lossy layers that allow the excitationenergy to penetrate further into the excitation region of a sample well.An enhancement of excitation energy may be a comparative reference to anembodiment that does not include structures to enhance the excitationenergy at an excitation region of a sample well.

The terms “about,” “approximately,” and “substantially” may be used inthe present disclosure to refer to a value, and are intended toencompass the referenced value plus and minus acceptable variations. Theamount of variation could be less than 5% in some embodiments, less than10% in some embodiments, and yet less than 20% in some embodiments. Inembodiments where an apparatus may function properly over a large rangeof values, e.g., a range including one or more orders of magnitude, theamount of variation could be a factor of two. For example, if anapparatus functions properly for a value ranging from 20 to 350,“approximately 80” may encompass values between 40 and 160.

The term “adjacent” may be used in the present disclosure to refer totwo elements arranged within close proximity to one another (e.g.,within a distance that is less than about one-fifth of a transverse orvertical dimension of a pixel). In some cases there may be interveningstructures or layers between adjacent elements. In some cases adjacentelements may be immediately adjacent to one another with no interveningstructures or elements.

The term “detect” may be used in the present disclosure to refer toreceiving an emission at a sensor from a sample well and producing atleast one electrical signal representative of or associated with theemission. The term “detect” may also be used in the present disclosureto refer to determining the presence of, or identifying a property of, aparticular sample or tag in the sample well based upon emission from thesample well.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the figures, described herein,are for illustration purposes only. It is to be understood that in someinstances various aspects of the invention may be shown exaggerated orenlarged to facilitate an understanding of the invention. In thedrawings, like reference characters generally refer to like features,functionally similar and/or structurally similar elements throughout thevarious figures. The drawings are not necessarily to scale, emphasisinstead being placed upon illustrating the principles of the teachings.The drawings are not intended to limit the scope of the presentteachings in any way.

FIG. 1-1 depicts emission wavelength spectra, according to someembodiments.

FIG. 1-2A depicts absorption wavelength spectra, according to someembodiments.

FIG. 1-2B depicts emission wavelength spectra, according to someembodiments.

FIG. 2-1 is a block diagram representation of an apparatus that may beused for rapid, mobile analysis of biological and chemical specimens,according to some embodiments.

FIG. 2-2 a schematic diagram of the relationship between pixels of thesensor chip and pixels of the assay chip, according to some embodiments.

FIG. 2-3 depicts components associated with a single pixel of the assaychip and a single pixel of the sensor chip, according to someembodiments.

FIG. 2-4 depicts a portion of the components of the instrument,according to some embodiments.

FIG. 3-1A is a top view of the assay chip and a chip holder frame,according to some embodiments.

FIG. 3-1B is a bottom view of the assay chip and the chip holder frame,according to some embodiments.

FIG. 3-1C depicts the assay chip and the chip holder frame, according tosome embodiments.

FIG. 3-2 depicts excitation energy incident on a sample well, accordingto some embodiments.

FIG. 3-3 illustrates attenuation of excitation energy along a samplewell that is formed as a zero-mode waveguide, according to someembodiments.

FIG. 3-4 depicts a sample well that includes a divot, which increasesexcitation energy at an excitation region associated with the samplewell in some embodiments.

FIG. 3-5 compares excitation intensities for sample wells with andwithout a divot, according to one embodiment.

FIG. 3-6 depicts a sample well and divot formed at a protrusion,according to some embodiments.

FIG. 3-7A depicts a sample well having tapered sidewalls, according tosome embodiments.

FIG. 3-7B depicts a sample well having curved sidewalls and a divot witha smaller transverse dimension, according to some embodiments.

FIG. 3-7C and FIG. 3-7D depict a sample well formed from surfaceplasmonic structures.

FIG. 3-7E depicts a sample well that includes anexcitation-energy-enhancing structure formed along sidewalls of thesample well, according to some embodiments.

FIG. 3-7F depicts a sample well formed in a multi-layer stack, accordingto some embodiments.

FIG. 3-8 illustrates surface coating formed on surfaces of a samplewell, according to some embodiments.

FIG. 3-9A through FIG. 3-9E depict structures associated with a lift-offprocess of forming a sample well, according to some embodiments.

FIG. 3-9F depicts a structure associated with an alternative lift-offprocess of forming a sample well, according to some embodiments.

FIG. 3-10A through FIG. 3-10D depict structures associated with a directetching process of forming a sample well, according to some embodiments.

FIG. 3-11 depicts a sample well that may be formed in multiple layersusing a lift-off process or a direct etching process, according to someembodiments.

FIG. 3-12 depicts a structure associated with an etching process thatmay be used to form a divot, according to some embodiments.

FIG. 3-13A through FIG. 3-13C depict structures associated with analternative process of forming a divot, according to some embodiments.

FIG. 3-14A through FIG. 3-14D depict structures associated with aprocess for depositing an adherent and passivating layers, according tosome embodiments.

FIG. 3-15 depicts a structure associated with a process for depositingan adherent centrally within a sample well, according to someembodiments.

FIG. 4-1A and FIG. 4-1B depict a surface-plasmon structure, according tojust one embodiment.

FIG. 4-1C depicts a surface-plasmon structure formed adjacent a samplewell, according to some embodiments.

FIG. 4-1D and FIG. 4-1E depict surface-plasmon structures formed in asample well, according to some embodiments.

FIG. 4-2A through FIG. 4-2C depict examples of periodic surface-plasmonstructures, according to some embodiments.

FIG. 4-2D depicts a numerical simulation of excitation energy at asample well-formed adjacent a periodic surface-plasmon structure,according to some embodiments.

FIG. 4-2E through FIG. 4-2G depict periodic surface-plasmon structures,according to some embodiments.

FIG. 4-2H and FIG. 4-2I depict a nano-antenna comprising surface-plasmonstructures, according to some embodiments.

FIG. 4-3A through FIG. 4-3E depict structures associated with processsteps for forming a surface-plasmon structure, according to someembodiments.

FIG. 4-4A through FIG. 4-4G depict structures associated with processsteps for forming a surface-plasmon structure and self-aligned samplewell, according to some embodiments.

FIG. 4-5A through FIG. 4-5E depict structures associated with processsteps for forming a surface-plasmon structure and self-aligned samplewell, according to some embodiments.

FIG. 4-6A depicts a thin lossy film formed adjacent a sample well,according to some embodiments.

FIG. 4-6B and FIG. 4-6C depict results from numerical simulations ofexcitation energy in the vicinity of a sample well and thin lossy film,according to some embodiments.

FIG. 4-6D depicts a thin lossy film spaced from a sample well, accordingto some embodiments.

FIG. 4-6E depicts a thin lossy film stack formed adjacent a sample well,according to some embodiments.

FIG. 4-7A illustrates a reflective stack that may be used to form aresonant cavity adjacent a sample well, according to some embodiments.

FIG. 4-7B depicts a dielectric structure that may be used to concentrateexcitation energy at a sample well, according to some embodiments.

FIG. 4-7C and FIG. 4-7D depict a photonic bandgap structure that may bepatterned adjacent a sample well, according to some embodiments.

FIG. 4-8A through FIG. 4-8G depict structures associated with processsteps for forming dielectric structures and a self-aligned sample well,according to some embodiments.

FIG. 4-9A and FIG. 4-9B depict structures for coupling excitation energyto a sample via a non-radiative process, according to some embodiments.

FIG. 4-9C depicts a structure for coupling excitation energy to a sampleby multiple non-radiative processes, according to some embodiments.

FIG. 4-9D depicts a structure that incorporates one or moreenergy-converting particles to couple excitation energy to a sample viaa radiative or non-radiative process, according to some embodiments.

FIG. 4-9E depicts spectra associated with down conversion of excitationenergy to a sample, according to some embodiments.

FIG. 4-9F depicts spectra associated with up conversion of excitationenergy to a sample, according to some embodiments.

FIG. 5-1 depicts a concentric, plasmonic circular grating, according tosome embodiments.

FIG. 5-2 depicts a spiral plasmonic grating, according to someembodiments.

FIG. 5-3 depict emission spatial distribution patterns from aconcentric, plasmonic circular grating, according to some embodiments.

FIG. 5-4A through FIG. 5-4B depict plasmonic nano-antennas, according tosome embodiments.

FIG. 5-5A through FIG. 5-5B depict plasmonic nano-antennas, according tosome embodiments.

FIG. 5-6A depicts refractive optics of the assay chip, according to someembodiments.

FIG. 5-6B depicts Fresnel lenses of the assay chip, according to someembodiments.

FIG. 6-1 depicts microscopy components of the instrument, according tosome embodiments.

FIG. 6-2A depicts far-field spectral sorting elements of the sensorchip, according to some embodiments.

FIG. 6-2B depicts far-field filtering elements of the sensor chip,according to some embodiments.

FIG. 6-3A and FIG. 6-3B depict thin lossy films of the sensor chip,according to some embodiments.

FIG. 6-4 depicts the optical block of the instrument, according to someembodiments.

FIG. 7-1A depicts, in elevation view, a sensor within a pixel of asensor chip, according to some embodiments.

FIG. 7-1B depicts a bulls-eye sensor having two separate and concentricactive areas, according to some embodiments.

FIG. 7-1C depicts a stripe sensor having four separate active areas,according to some embodiments.

FIG. 7-1D depicts a quad sensor having four separate active areas,according to some embodiments.

FIG. 7-1E depicts an arc-segment sensor having four separate activeareas, according to some embodiments.

FIG. 7-1F depicts a stacked-segment sensor, according to someembodiments.

FIG. 7-2A depicts an emission distribution from the sorting elements forenergy emitted at a first wavelength, according to some embodiments.

FIG. 7-2B depicts a radiation pattern received by a bulls-eye sensorcorresponding to the emission distribution depicted in FIG. 7-2A,according to some embodiments.

FIG. 7-2C depicts an emission distribution from the sorting elements forenergy emitted at a second wavelength, according to some embodiments.

FIG. 7-2D depicts a radiation pattern received by a bulls-eye sensorcorresponding to the emission distribution depicted in FIG. 7-2C,according to some embodiments.

FIG. 7-2E represents results from a numerical simulation of signaldetection for a bulls-eye sensor having two active areas for a firstemission wavelength from a sample, according to some embodiments.

FIG. 7-2F represents results from a numerical simulation of signaldetection for the bulls-eye sensor associated with FIG. 7-2E for asecond emission wavelength from a sample, according to some embodiments.

FIG. 7-2G represents results from a numerical simulation of signaldetection for the bulls-eye sensor associated with FIG. 7-2E for a thirdemission wavelength from a sample, according to some embodiments.

FIG. 7-2H represents results from a numerical simulation of signaldetection for the bulls-eye sensor associated with FIG. 7-2E for afourth emission wavelength from a sample, according to some embodiments.

FIG. 7-2I represents results from a numerical simulation of signaldetection for a bulls-eye sensor having four active areas for a firstemission wavelength from a sample, according to some embodiments.

FIG. 7-2J represents results from a numerical simulation of signaldetection for the bulls-eye sensor associated with FIG. 7-2I for asecond emission wavelength from a sample, according to some embodiments.

FIG. 7-3A depicts circuitry on an instrument that may be used to readsignals from a sensor comprising two active areas, according to someembodiments.

FIG. 7-3B depicts a three-transistor circuit that may be included at asensor chip for signal accumulation and read-out, according to someembodiments.

FIG. 7-3C depicts circuitry on an instrument that may be used to readsignals from a sensor comprising four active areas, according to someembodiments.

FIG. 7-4A depicts temporal emission characteristics for two differentemitters that may be used for sample analysis, according to someembodiments.

FIG. 7-4B depicts temporal evolution of an excitation source andluminescence from a sample, according to some embodiments.

FIG. 7-4C illustrates time-delay sampling, according to someembodiments.

FIG. 7-4D depicts temporal emission characteristics for two differentemitters, according to some embodiments.

FIG. 7-4E depicts voltage dynamics at a charge-accumulation node of asensor, according to some embodiments.

FIG. 7-4F depicts a double read of a sensor segment without reset,according to some embodiments.

FIG. 7-4G and FIG. 7-4H illustrate first and second read signal levelsassociated with two emitters having temporally-distinct emissioncharacteristics, according to some embodiments.

FIG. 8-1A and FIG. 8-1B depict spectral excitation bands of excitationsources, according to some embodiments.

FIG. 9-1 depicts a method of operation of a compact apparatus that maybe used for rapid, mobile analysis of biological and chemical specimens,according to some embodiments.

FIG. 9-2 depicts a calibration procedure, according to some embodiments.

FIG. 9-3 depicts a data-analysis procedure, according to someembodiments.

FIG. 10 depicts a computing environment, according to some embodiments.

The features and advantages of embodiments of the present applicationwill become more apparent from the detailed description set forth belowwhen taken in conjunction with the drawings.

DETAILED DESCRIPTION I. Inventor's Recognition of the Problem andSolution Thereto

The inventors have recognized and appreciated that conventionalapparatuses for performing bioassays are large, expensive and requireadvanced laboratory techniques to perform. Many types of bioassaysdepend on the detection of single molecules in a specimen.Conventionally single molecule detection may require large, bulky lasersystems used to generate high intensity light needed for excitation ofmolecules. In addition, bulky optical components may be used to directthe laser light to the specimen and additional optical components may beused to direct luminescent light from the specimen to a sensor. Theseconventional optical components may require precise alignment andstabilization. The conventional laboratory equipment and trainingrequired to use this conventional equipment may result in complex,expensive bioassays.

The inventors have recognized and appreciated that there is a need for adevice that can simply and inexpensively analyze biological and/orchemical specimens to determine the identity of its constituent parts.An application of such device may be for sequencing a biomolecule, suchas a nucleic acid molecule or a polypeptide (e.g., protein) having aplurality of amino acids. A compact, high-speed apparatus for performingdetection and quantitation of single molecules or particles could reducethe cost of performing complex quantitative measurements of biologicaland/or chemical samples and rapidly advance the rate of biochemicaltechnological discoveries. Moreover, a cost-effective device that isreadily transportable could transform not only the way bioassays areperformed in the developed world, but provide people in developingregions, for the first time, ready access to essential diagnostic teststhat could dramatically improve their health and well-being. Forexample, in some embodiments, an apparatus for performing bioassays isused to perform diagnostic tests of biological samples, such as blood,urine and/or saliva. The apparatus may be used by individuals in theirhome, by a doctor in a remote clinics in developing countries or anyother location, such as rural doctors' offices. Such diagnostic testscan include the detection of biomolecules in a biological sample of asubject, such as a nucleic acid molecule or protein. In some examples,diagnostic tests include sequencing a nucleic acid molecule in abiological sample of a subject, such as sequencing of cell freedeoxyribonucleic acid molecules or expression products in a biologicalsample of the subject.

The term “nucleic acid,” as used herein, generally refers to a moleculecomprising one or more nucleic acid subunits. A nucleic acid may includeone or more subunits selected from adenosine (A), cytosine (C), guanine(G), thymine (T) and uracil (U), or variants thereof. In some examples,a nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA),or derivatives thereof. A nucleic acid may be single-stranded or doublestranded. A nucleic acid may be circular.

The term “nucleotide,” as used herein, generally refers to a nucleicacid subunit, which can include A, C, G, T or U, or variants or analogsthereof. A nucleotide can include any subunit that can be incorporatedinto a growing nucleic acid strand. Such subunit can be an A, C, G, T,or U, or any other subunit that is specific to one or more complementaryA, C, G, T or U, or complementary to a purine (i.e., A or G, or variantor analogs thereof) or a pyrimidine (i.e., C, T or U, or variant oranalogs thereof). A subunit can enable individual nucleic acid bases orgroups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, oruracil-counterparts thereof) to be resolved.

A nucleotide generally includes a nucleoside and at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more phosphate (PO3) groups. A nucleotide can includea nucleobase, a five-carbon sugar (either ribose or deoxyribose), andone or more phosphate groups. Ribonucleotides are nucleotides in whichthe sugar is ribose. Deoxyribonucleotides are nucleotides in which thesugar is deoxyribose. A nucleotide can be a nucleoside monophosphate ora nucleoside polyphosphate. A nucleotide can be a deoxyribonucleosidepolyphosphate, such as, e.g., a deoxyribonucleoside triphosphate, whichcan be selected from deoxyadenosine triphosphate (dATP), deoxycytidinetriphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyuridinetriphosphate (dUTP) and deoxythymidine triphosphate (dTTP) dNTPs, thatinclude detectable tags, such as luminescent tags or markers (e.g.,fluorophores).

A nucleoside polyphosphate can have ‘n’ phosphate groups, where ‘n’ is anumber that is greater than or equal to 2, 3, 4, 5, 6, 7, 8, 9, or 10.Examples of nucleoside polyphosphates include nucleoside diphosphate andnucleoside triphosphate. A nucleotide can be a terminal phosphatelabeled nucleoside, such as a terminal phosphate labeled nucleosidepolyphosphate. Such label can be a luminescent (e.g., fluorescent orchemiluminescent) label, a fluorogenic label, a colored label, achromogenic label, a mass tag, an electrostatic label, or anelectrochemical label. A label (or marker) can be coupled to a terminalphosphate through a linker. The linker can include, for example, atleast one or a plurality of hydroxyl groups, sulfhydryl groups, aminogroups or haloalkyl groups, which may be suitable for forming, forexample, a phosphate ester, a thioester, a phosphoramidate or an alkylphosphonate linkage at the terminal phosphate of a natural or modifiednucleotide. A linker can be cleavable so as to separate a label from theterminal phosphate, such as with the aid of a polymerization enzyme.Examples of nucleotides and linkers are provided in U.S. Pat. No.7,041,812, which is entirely incorporated herein by reference.

The term “polymerase,” as used herein, generally refers to any enzyme(or polymerizing enzyme) capable of catalyzing a polymerizationreaction. Examples of polymerases include, without limitation, a nucleicacid polymerase, a transcriptase or a ligase. A polymerase can be apolymerization enzyme.

The term “genome” generally refers to an entirety of an organism'shereditary information. A genome can be encoded either in DNA or in RNA.A genome can comprise coding regions that code for proteins as well asnon-coding regions. A genome can include the sequence of all chromosomestogether in an organism. For example, the human genome has a total of 46chromosomes. The sequence of all of these together constitutes the humangenome.

The present disclosure provides devices, systems and methods fordetecting biomolecules or subunits thereof, such as nucleic acidmolecules. Such detection can include sequencing. A biomolecule may beextracted from a biological sample obtained from a subject. Thebiological sample may be extracted from a bodily fluid or tissue of thesubject, such as breath, saliva, urine or blood (e.g., whole blood orplasma). The subject may be suspected of having a health condition, suchas a disease (e.g., cancer). In some examples, one or more nucleic acidmolecules are extracted from the bodily fluid or tissue of the subject.The one or more nucleic acids may be extracted from one or more cellsobtained from the subject, such as part of a tissue of the subject, orobtained from a cell-free bodily fluid of the subject, such as wholeblood.

A biological sample may be processed in preparation for detection (e.g.,sequencing). Such processing can include isolation and/or purificationof the biomolecule (e.g., nucleic acid molecule) from the biologicalsample, and generation of more copies of the biomolecule. In someexamples, one or more nucleic acid molecules are isolated and purifiedform a bodily fluid or tissue of the subject, and amplified throughnucleic acid amplification, such as polymerase chain reaction (PCR).Then, the one or more nucleic acids molecules or subunits thereof can beidentified, such as through sequencing.

Sequencing can include the determination of individual subunits of atemplate biomolecule (e.g., nucleic acid molecule) by synthesizinganother biomolecule that is complementary or analogous to the template,such as by synthesizing a nucleic acid molecule that is complementary toa template nucleic acid molecule and identifying the incorporation ofnucleotides with time (i.e., sequencing by synthesis). As analternative, sequencing can include the direct identification ofindividual subunits of the biomolecule.

During sequencing, signals indicative of individual subunits of abiomolecule may be collected in memory and processed in real time or ata later point in time to determine a sequence of the biomolecule. Suchprocessing can include a comparison of the signals to reference signalsthat enable the identification of the individual subunits, which in somecases yields reads. Reads may be sequences of sufficient length (e.g.,at least about 30 base pairs (bp)) that can be used to identify a largersequence or region, e.g., that can be aligned to a location on achromosome or genomic region or gene.

Sequence reads can be used to reconstruct a longer region of a genome ofa subject (alignment). Reads can be used to reconstruct chromosomalregions, whole chromosomes, or the whole genome. Sequence reads or alarger sequence generated from such reads can be used to analyze agenome of a subject, such as identify variants or polymorphisms.Examples of variants include, but are not limited to, single nucleotidepolymorphisms (SNPs) including tandem SNPs, small-scale multi-basedeletions or insertions, also referred to as indels or deletioninsertion polymorphisms or DIPs), Multi-Nucleotide Polymorphisms (MNPs),Short Tandem Repeats (STRs), deletions, including microdeletions,insertions, including microinsertions, structural variations, includingduplications, inversions, translocations, multiplications, complexmulti-site variants, copy number variations (CNV). Genomic sequences cancomprise combinations of variants. For example, genomic sequences canencompass the combination of one or more SNPs and one or more CNVs.

Individual subunits of biomolecules may be identified using markers. Insome examples, luminescent markers are used to identified individualsubunits of biomolecules. Some embodiments use luminescent markers (alsoreferred to herein as “markers”), which may be exogenous or endogenousmarkers. Exogenous markers may be external luminescent markers used as areporter and/or tag for luminescent labeling. Examples of exogenousmarkers may include, but are not limited to, fluorescent molecules,fluorophores, fluorescent dyes, fluorescent stains, organic dyes,fluorescent proteins, species that participate in fluorescence resonanceenergy transfer (FRET), enzymes, and/or quantum dots. Other exogenousmarkers are known in the art. Such exogenous markers may be conjugatedto a probe or functional group (e.g., molecule, ion, and/or ligand) thatspecifically binds to a particular target or component. Attaching anexogenous tag or reporter to a probe allows identification of the targetthrough detection of the presence of the exogenous tag or reporter.Examples of probes may include proteins, nucleic acid (e.g., DNA, RNA)molecules, lipids and antibody probes. The combination of an exogenousmarker and a functional group may form any suitable probes, tags, and/orlabels used for detection, including molecular probes, labeled probes,hybridization probes, antibody probes, protein probes (e.g.,biotin-binding probes), enzyme labels, fluorescent probes, fluorescenttags, and/or enzyme reporters.

Although the present disclosure makes reference to luminescent markers,other types of markers may be used with devices, systems and methodsprovided herein. Such markers may be mass tags, electrostatic tags, orelectrochemical labels.

While exogenous markers may be added to a sample, endogenous markers maybe already part of the sample. Endogenous markers may include anyluminescent marker present that may luminesce or “autofluoresce” in thepresence of excitation energy. Autofluorescence of endogenousfluorophores may provide for label-free and noninvasive labeling withoutrequiring the introduction of exogenous fluorophores. Examples of suchendogenous fluorophores may include hemoglobin, oxyhemoglobin, lipids,collagen and elastin crosslinks, reduced nicotinamide adeninedinucleotide (NADH), oxidized flavins (FAD and FMN), lipofuscin,keratin, and/or prophyrins, by way of example and not limitation.

While some embodiments may be directed to diagnostic testing bydetecting single molecules in a specimen, the inventors have alsorecognized that the single molecule detection capabilities of thepresent disclosure may be used to perform polypeptide (e.g., protein)sequencing or nucleic acid (e.g., DNA, RNA) sequencing of one or morenucleic acid segments of, for example, genes. Nucleic acid sequencingtechnologies may vary in the methods used to determine the nucleic acidsequence as well as in the rate, read length, and incidence of errors inthe sequencing process. For example, some nucleic acid sequencingmethods are based on sequencing by synthesis, in which the identity of anucleotide is determined as the nucleotide is incorporated into a newlysynthesized strand of nucleic acid that is complementary to the targetnucleic acid.

During sequencing, a polymerizing enzyme may couple (e.g., attach) to apriming location of a target nucleic acid molecule. The priming locationcan be a primer that is complementary to the target nucleic acidmolecule. As an alternative the priming location is a gap or nick thatis provided within a double stranded segment of the target nucleic acidmolecule. A gap or nick can be from 0 to at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, or 40 nucleotides in length. A nick can provide abreak in one strand of a double stranded sequence, which can provide apriming location for a polymerizing enzyme, such as, for example, astrand displacing polymerase enzyme.

In some cases, a sequencing primer can be annealed to a target nucleicacid molecule that may or may not be immobilized to a solid support,such as a sample well. In some embodiments, a sequencing primer may beimmobilized to a solid support and hybridization of the target nucleicacid molecule also immobilizes the target nucleic acid molecule to thesolid support. Via the action of an enzyme (e.g., a polymerase) capableof adding or incorporating a nucleotide to the primer, nucleotides canbe added to the primer in 5′ to 3′, template bound fashion. Suchincorporation of nucleotides to a primer (e.g., via the action of apolymerase) can generally be referred to as a primer extension reaction.Each nucleotide can be associated with a detectable tag that can bedetected and used to determine each nucleotide incorporated into theprimer and, thus, a sequence of the newly synthesized nucleic acidmolecule. Via sequence complementarity of the newly synthesized nucleicacid molecule, the sequence of the target nucleic acid molecule can alsobe determined. In some cases, annealing of a sequencing primer to atarget nucleic acid molecule and incorporation of nucleotides to thesequencing primer can occur at similar reaction conditions (e.g., thesame or similar reaction temperature) or at differing reactionconditions (e.g., different reaction temperatures). Moreover, somesequencing by synthesis methods can include the presence of a populationof target nucleic acid molecules (e.g., copies of a target nucleic acid)and/or a step of amplification of the target nucleic acid to achieve apopulation of target nucleic acids.

Embodiments are capable of sequencing single nucleic acid molecules withhigh accuracy and long read lengths, such as an accuracy of at leastabout 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%,99.99%, 99.999%, or 99.9999%, and/or read lengths greater than or equalto about 10 base pairs (bp), 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500bp, 1000 bp, 10,000 bp, 20,000 bp, 30,000 bp, 40,000 bp, 50,000 bp, or100,000 bp. In some embodiments, the target nucleic acid molecule usedin single molecule sequencing is a single stranded target nucleic acid(e.g., deoxyribonucleic acid (DNA), DNA derivatives, ribonucleic acid(RNA), RNA derivatives) template that is added or immobilized to asample well containing at least one additional component of a sequencingreaction (e.g., a polymerase such as, a DNA polymerase, a sequencingprimer) immobilized or attached to a solid support such as the bottom ofthe sample well. The target nucleic acid molecule or the polymerase canbe attached to a sample wall, such as at the bottom of the sample welldirectly or through a linker. The sample well can also contain any otherreagents needed for nucleic acid synthesis via a primer extensionreaction, such as, for example suitable buffers, co-factors, enzymes(e.g., a polymerase) and deoxyribonucleoside polyphosphates, such as,e.g., deoxyribonucleoside triphosphates, including deoxyadenosinetriphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosinetriphosphate (dGTP), deoxyuridine triphosphate (dUTP) and deoxythymidinetriphosphate (dTTP) dNTPs, that include luminescent tags, such asfluorophores. Each class of dNTPs (e.g. adenine-containing dNTPs (e.g.,dATP), cytosine-containing dNTPs (e.g., dCTP), guanine-containing dNTPs(e.g., dGTP), uracil-containing dNTPs (e.g., dUTPs) andthymine-containing dNTPs (e.g., dTTP)) is conjugated to a distinctluminescent tag such that detection of light emitted from the tagindicates the identity of the dNTP that was incorporated into the newlysynthesized nucleic acid. Emitted light from the luminescent tag can bedetected and attributed to its appropriate luminescent tag (and, thus,associated dNTP) via any suitable device and/or method, including suchdevices and methods for detection described elsewhere herein. Theluminescent tag may be conjugated to the dNTP at any position such thatthe presence of the luminescent tag does not inhibit the incorporationof the dNTP into the newly synthesized nucleic acid strand or theactivity of the polymerase. In some embodiments, the luminescent tag isconjugated to the terminal phosphate (the gamma phosphate) of the dNTP.

The single-stranded target nucleic acid template can be contacted with asequencing primer, dNTPs, polymerase and other reagents necessary fornucleic acid synthesis. In some embodiments, all appropriate dNTPs canbe contacted with the single-stranded target nucleic acid templatesimultaneously (e.g., all dNTPs are simultaneously present) such thatincorporation of dNTPs can occur continuously. In other embodiments, thedNTPs can be contacted with the single-stranded target nucleic acidtemplate sequentially, where the single-stranded target nucleic acidtemplate is contacted with each appropriate dNTP separately, withwashing steps in between contact of the single-stranded target nucleicacid template with differing dNTPs. Such a cycle of contacting thesingle-stranded target nucleic acid template with each dNTP separatelyfollowed by washing can be repeated for each successive base position ofthe single-stranded target nucleic acid template to be identified.

The sequencing primer anneals to the single-stranded target nucleic acidtemplate and the polymerase consecutively incorporates the dNTPs (orother deoxyribonucleoside polyphosphate) to the primer via thesingle-stranded target nucleic acid template. The unique luminescent tagassociated with each incorporated dNTP can be excited with theappropriate excitation light during or after incorporation of the dNTPto the primer and its emission can be subsequently detected, using, anysuitable device(s) and/or method(s), including devices and methods fordetection described elsewhere herein. Detection of a particular emissionof light can be attributed to a particular dNTP incorporated. Thesequence obtained from the collection of detected luminescent tags canthen be used to determine the sequence of the single-stranded targetnucleic acid template via sequence complementarity.

While the present disclosure makes reference to dNTPs, devices, systemsand methods provided herein may be used with various types ofnucleotides, such as ribonucleotides and deoxyribonucleotides (e.g.,deoxyribonucleoside polyphophates with at least 4, 5, 6, 7, 8, 9, or 10phosphate groups). Such ribonucleotides and deoxyribonucleotides caninclude various types of tags (or markers) and linkers.

Signals emitted upon the incorporation of nucleosides can be stored inmemory and processed at a later point in time to determine the sequenceof the target nucleic acid template. This may include comparing thesignals to a reference signals to determine the identities of theincorporated nucleosides as a function of time. Alternative or inaddition to, signal emitted upon the incorporation of nucleoside can becollected and processed in real time (i.e., upon nucleosideincorporation) to determine the sequence of the target nucleic acidtemplate in real time.

Nucleic acid sequencing of a plurality of single-stranded target nucleicacid templates may be completed where multiple sample wells areavailable, as is the case in devices described elsewhere herein. Eachsample well can be provided with a single-stranded target nucleic acidtemplate and a sequencing reaction can be completed in each sample well.Each of the sample wells may be contacted with the appropriate reagents(e.g., dNTPs, sequencing primers, polymerase, co-factors, appropriatebuffers, etc.) necessary for nucleic acid synthesis during a primerextension reaction and the sequencing reaction can proceed in eachsample well. In some embodiments, the multiple sample wells arecontacted with all appropriate dNTPs simultaneously. In otherembodiments, the multiple sample wells are contacted with eachappropriate dNTP separately and each washed in between contact withdifferent dNTPs. Incorporated dNTPs can be detected in each sample welland a sequence determined for the single-stranded target nucleic acid ineach sample well as is described above.

Embodiments directed towards single molecule nucleic acid sequencing mayuse any polymerase that is capable of synthesizing a nucleic acidcomplementary to a target nucleic acid molecule. Examples of polymerasesinclude, but are not limited to, a DNA polymerase, an RNA polymerase, athermostable polymerase, a wild-type polymerase, a modified polymerase,E. coli DNA polymerase I, T7 DNA polymerase, bacteriophage T4 DNApolymerase φ29 (psi29) DNA polymerase, Taq polymerase, Tth polymerase,Tli polymerase, Pfu polymerase, Pwo polymerase, VENT polymerase,DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Ssopolymerase, Poc polymerase, Pab polymerase, Mth polymerase, ES4polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tmapolymerase, Tca polymerase, Tih polymerase, Tfi polymerase, Platinum Taqpolymerases, Tbr polymerase, Tfl polymerase, Tth polymerase, Pfutubopolymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bstpolymerase, Sac polymerase, Klenow fragment, polymerase with 3′ to 5′exonuclease activity, and variants, modified products and derivativesthereof. In some embodiments, the polymerase is a single subunitpolymerase. In some embodiments, the polymerase is a polymerase withhigh processivity. Polymerase processivity generally refers to thecapability of a polymerase to consecutively incorporate dNTPs into anucleic acid template without releasing the nucleic acid template. Uponbase pairing between a nucleobase of a target nucleic acid and thecomplementary dNTP, the polymerase incorporates the dNTP into the newlysynthesized nucleic acid strand by forming a phosphodiester bond betweenthe 3′ hydroxyl end of the newly synthesized strand and the alphaphosphate of the dNTP. In examples in which the luminescent tagconjugated to the dNTP is a fluorophore, its presence is signaled byexcitation and a pulse of emission is detected during or after the stepof incorporation. For detection labels that are conjugated to theterminal (gamma) phosphate of the dNTP, incorporation of the dNTP intothe newly synthesized strand results in release the beta and gammaphosphates and the detection label, which is free to diffuse in thesample well, resulting in a decrease in emission detected from thefluorophore.

Embodiments directed toward single molecule RNA sequencing may use anyreverse transcriptase that is capable of synthesizing complementary DNA(cDNA) from an RNA template. In such embodiments, a reversetranscriptase can function in a manner similar to polymerase in thatcDNA can be synthesized from an RNA template via the incorporation ofdNTPs to a reverse transcription primer annealed to an RNA template. ThecDNA can then participate in a sequencing reaction and its sequencedetermined as described above. The determined sequence of the cDNA canthen be used, via sequence complementarity, to determine the sequence ofthe original RNA template. Examples of reverse transcriptases includeMoloney Murine Leukemia Virus reverse transcriptase (M-MLV), avianmyeloblastosis virus (AMV) reverse transcriptase, human immunodeficiencyvirus reverse transcriptase (HIV-1) and telomerase reversetranscriptase.

Having recognized the need for simple, less complex apparatuses forperforming single molecule detection and/or nucleic acid sequencing, theinventors have conceived of techniques for detecting single moleculesusing sets of luminescent tags to label different molecules. Such singlemolecules may be nucleotides or amino acids having tags. Tags may bedetected while bound to single molecules, upon release from the singlemolecules, or while bound to and upon release from the single molecules.In some examples, tags are luminescent tags. Each luminescent tag in aselected set is associated with a respective molecule. For example, aset of four tags may be used to “label” the nucleobases present inDNA—each tag of the set being associated with a different nucleobase,e.g., a first tag being associated with adenine (A), a second tag beingassociated with cytosine (C), a third tag being associated with guanine(G), and a fourth tag being associated with thymine (T). Moreover, eachof the luminescent tags in the set of tags has different properties thatmay be used to distinguish a first tag of the set from the other tags inthe set. In this way, each tag is uniquely identifiable using one ormore of these distinguishing characteristics. By way of example and notlimitation, the characteristics of the tags that may be used todistinguish one tag from another may include the emission energy and/orwavelength of the light that is emitted by the tag in response toexcitation energy and/or the wavelength of the excitation light that isabsorbed by a particular tag to place the tag in an excited state.

Embodiments may use any suitable combination of tag characteristics todistinguish a first tag in a set of tags from the other tags in the sameset. For example, some embodiments may use only the wavelength of theemission light from the tags to identify the tags. In such embodiments,each tag in a selected set of tags has a different peak emissionwavelength from the other tags in the set and the luminescent tags areall excited by light from a single excitation source. FIG. 1-1illustrates the emission spectra from four luminescent tags according toan embodiment where the four tags exhibit their respective intensitypeak at different emission wavelengths, referred to herein as the tag's“peak emission wavelength.” A first emission spectrum 1-101 from a firstluminescent tag has a peak emission wavelength at λ1, a second emissionspectrum 1-102 from a second luminescent tag has a peak emissionwavelength at λ2, a third emission spectrum 1-103 from a thirdluminescent tag has a peak emission wavelength at λ3, and a fourthemission spectrum 1-104 from a fourth luminescent tag has a peakemission wavelength at λ4. In this embodiment, the emission peaks of thefour luminescent tags may have any suitable values that satisfy therelation λ1<λ2<λ3<λ4. The four emission spectra may or may not overlap.However, if the emission spectra of two or more tags overlap, it isdesirable to select a luminescent tag set such that one tag emitssubstantially more light than any other tag at each respective peakwavelength. In this embodiment, the excitation wavelength at which eachof the four tags maximally absorbs light from the excitation source issubstantially the same, but that need not be the case. Using the abovetag set, four different molecules may be labeled with a respective tagfrom the tag set, the tags may be excited using a single excitationsource, and the tags can be distinguished from one another by detectingthe emission wavelength of the tags using an optical system and sensors.While FIG. 1-1 illustrates four different tags, it should be appreciatedthat any suitable number of tags may be used.

Other embodiments may use both the wavelength of the emission light fromthe tags and the wavelength at which the tags absorb excitation light toidentify the tags. In such embodiments, each tag in a selected set oftags has a different combination of emission wavelength and excitationwavelength from the other tags in the set. Thus, some tags within aselected tag set may have the same emission wavelength, but be excitedby light of different wavelengths. Conversely, some tags within aselected tag set may have the same excitation wavelength, but emit lightat different wavelengths. FIG. 1-2A illustrates the emission spectrafrom four luminescent tags according to an embodiment where two of thetags have a first peak emission wavelength and the other two tags have asecond peak emission wavelength. A first emission spectrum 1-105 from afirst luminescent tag has a peak emission wavelength at λ1, a secondemission spectrum 1-106 from a second luminescent tag also has a peakemission wavelength at λ1, a third emission spectrum 1-107 from a thirdluminescent tag has a peak emission wavelength at λ2, and a fourthemission spectrum 1-108 from a fourth luminescent tag also has a peakemission wavelength at λ2. In this embodiment, the emission peaks of thefour luminescent tags may have any suitable values that satisfy therelation λ1<λ2. FIG. 1-2B illustrates the absorption spectra from thefour luminescent tags, where two of the tags have a first peakabsorption wavelength and the other two tags have a second peakabsorption wavelength. A first absorption spectrum 1-109 for the firstluminescent tag has a peak absorption wavelength at λ3, a secondabsorption spectrum 1-110 for the second luminescent tag has a peakabsorption wavelength at λ4, a third absorption spectrum 1-111 for thethird luminescent tag has a peak absorption wavelength at λ3, and afourth absorption spectrum 1-112 for the fourth luminescent tag has apeak absorption wavelength at λ4. Note that the tags that share anemission peak wavelength in FIG. 1-2A do not share an absorption peakwavelength in FIG. 1-2B. Using such a tag set allows distinguishingbetween four tags even when there are only two emission wavelengths forthe four dyes. This is possible using two excitation sources that emitat different wavelengths or a single excitation source capable ofemitting at multiple wavelengths. If the wavelength of the excitationlight is known for each detected emission event, then it can bedetermined which tag was present. The excitation source(s) may alternatebetween a first excitation wavelength and a second excitationwavelength, which is referred to as interleaving. Alternatively, two ormore pulses of the first excitation wavelength may be used followed bytwo or more pulses of the second excitation wavelength.

While not illustrated in the figures, other embodiments may determinethe identity of a luminescent tag based on the absorption frequencyalone. Such embodiments are possible if the excitation light can betuned to specific wavelengths that match the absorption spectrum of thetags in a tag set. In such embodiments, the optical system and sensorused to direct and detect the light emitted from each tag do not need tobe capable of detecting the wavelength of the emitted light. This may beadvantageous in some embodiments because it reduces the complexity ofthe optical system and sensors because detecting the emission wavelengthis not required in such embodiments.

As discussed above, the inventors have recognized and appreciated theneed for being able to distinguish different optical (e.g., luminescent)tags from one another using various characteristics of the tags. Thetype of characteristics used to determine the identity of a tag impactthe physical device used to perform this analysis. The presentapplication discloses several embodiments of an apparatus, device,instrument and methods for performing these different techniques.

Briefly, the inventors have recognized and appreciated that a pixelatedsensor device with a relatively large number of pixels (e.g., hundreds,thousands, millions or more) that allows for the detection of aplurality of individual molecules or particles in parallel. Such singlemolecules may be nucleotides or amino acids having tags. Tags may bedetected while bound to single molecules, upon release from the singlemolecules, or while bound to and upon release from the single molecules.In some examples, tags are luminescent tags. The molecules may be, byway of example and not limitation, proteins and/or nucleic acids (e.g.,DNA, RNA). Moreover, a high-speed device that can acquire data at morethan one hundred frames per second allows for the detection and analysisof dynamic processes or changes that occur over time within the samplebeing analyzed.

The inventors have recognized and appreciated that a low-cost,single-use disposable assay chip may be used in connection with aninstrument that includes an excitation light source, optics, and a lightsensor to measure an optical signal (e.g., luminescent light) emittedfrom biological samples. Using a low-cost assay chip reduces the cost ofperforming a given bioassay. A biological sample is placed onto theassay chip and, upon completion of a single bioassay, may be discarded.In some embodiments, more than one type of sample may be analyzedsimultaneously, in parallel, by placing multiple samples on differentportions of the assay chip at the same time. The assay chip interfaceswith the more expensive, multi-use instrument, which may be usedrepeatedly with many different disposable assay chips. A low-cost assaychip that interfaces with a compact, portable instrument may be usedanywhere in the world, without the constraint of high-cost biologicallaboratories requiring laboratory expertise to analyze samples. Thus,automated bioanalytics may be brought to regions of the world thatpreviously could not perform quantitative analysis of biologicalsamples. For example, blood tests for infants may be performed byplacing a blood sample on a disposable assay chip, placing thedisposable assay chip into the small, portable instrument for analysis,and processing the results by a computer that connects to the instrumentfor immediate review by a user. The data may also be transmitted over adata network to a remote location to be analyzed, and/or archived forsubsequent clinical analyses. Alternatively, the instrument may includeone or more processors for analyzing the data obtained from the sensorsof the instrument.

Various embodiments are described in more detail below.

II. Overview of the Apparatus According to Some Embodiments

A schematic overview of the apparatus 2-100 is illustrated in FIG. 2-1.The system comprises both an assay chip 2-110 and an instrument 2-120comprising an excitation source 2-121 and at least one sensor 2-122. Theassay chip 2-110 interfaces with the instrument 2-120 using any suitableassay chip interface. For example, the assay chip interface of theinstrument 2-120 may include a socket (not illustrated) for receivingthe assay chip 2-110 and holding it in precise optical alignment withthe excitation source 2-110 and the at least one sensor 2-122. Theexternal excitation source 2-121 in the instrument 2-120 is configuredto provide excitation energy to the assay chip 2-110 for the purpose ofexciting a sample in the sample well 2-111 of the assay chip 2-110. Insome embodiments, the assay chip 2-110 has multiple pixels, the samplewell 2-111 of each pixel configured to receive a sample used in ananalysis independent from the other pixels. Each pixel of the assay chip2-110 comprises a sample well 2-211 for receiving, retaining andanalyzing a sample from the specimen being analyzed. Such pixels may bereferred to as “passive source pixels” since the pixels receiveexcitation energy from an excitation source separate from the pixel. Insome embodiments, there is a pixel in the instrument 2-120 correspondingto each pixel present on the assay chip 2-110. Each pixel of theinstrument 2-120 comprises at least one sensor for detecting emissionenergy emitted by the sample in response to the sample being illuminatedwith excitation energy from the excitation source 2-121. In someembodiments, each sensor may include multiple sub-sensors, eachsub-sensor configured to detect a different wavelength of emissionenergy from the sample. While more than one sub-sensor may detectemission energy of a certain wavelength, each sub-sensor may detect adifferent wavelength band of emission energy.

In some embodiments, optical elements for guiding and couplingexcitation energy from the excitation source 2-121 to the sample well2-111 are located both on the assay chip 2-110 and the instrument 2-120,as represented by arrow 2-101 in FIG. 2-1. Such source-to-well elementsmay include mirrors, lenses, dielectric coatings and beam combinerslocated on the instrument 2-120 to couple excitation energy to the assaychip 2-110 and lenses, plasmonic elements and dielectric coatings on theassay chip 1-110 to direct the excitation energy received from theinstrument 2-120 to the sample well 2-111. Additionally, in someembodiments, optical elements for guiding emission energy from thesample well 2-111 to the sensor 2-122 are located on the assay chip2-110 and the instrument 2-120, as represented by arrow 2-102 in FIG.2-1. Such well-to-sample elements may include may include lenses,plasmonic elements and dielectric coatings located on the assay chip2-110 to direct emission energy from the assay chip 2-110 to theinstrument 2-120 and lenses, mirrors, dielectric coatings, filters anddiffractive optics on the instrument 1-120 to direct the emission energyreceived from the assay chip 2-110 to the sensor 2-111. In someembodiments, a single component may play a role in both in couplingexcitation energy to a sample well and delivering emission energy fromthe sample well to the sensor.

In some embodiments, the assay chip 2-110 comprises a plurality ofpixels, each pixel associated with its own individual sample well 2-111and its own associated sensor 2-122 on the instrument 2-120. Theplurality of pixels may be arranged in an array and may have anysuitable number of pixels. For example, the assay chip may includeapproximately 1,000 pixels, 10,000 pixels, approximately 100,000 pixels,approximately 1,000,000 pixels, approximately 10,000,000 pixels, orapproximately 100,000,000 pixels.

In some embodiments, the instrument 2-120 includes a sensor chipcomprising a plurality of sensors 2-122 arranged as a plurality ofpixels. Each pixel of the sensor chip corresponds to a pixel in theassay chip 2-110. The plurality of pixels may be arranged in an arrayand may have any suitable number of pixels. In some embodiments, thesensor chip has the same number of pixels as the assay chip 2-110. Forexample, the sensor chip may include approximately 10,000 pixels,approximately 100,000 pixels, approximately 1,000,000 pixels,approximately 10,000,000 pixels, or approximately 100,000,000 pixels.

The instrument 2-120 interfaces with the assay chip 2-110 through anassay chip interface (not shown). The assay chip interface may includecomponents to position and/or align the assay chip 2-110 to theinstrument 2-120 to improve coupling of the excitation energy from theexcitation source 2-121to the assay chip 2-110. In some embodiments,excitation source 2-121 includes multiple excitation sources that arecombined to deliver excitation energy to the assay chip 2-110. Themultiple excitation sources may be configured to produce multipleexcitation energies, corresponding to light of different wavelengths.

The instrument 2-120 includes a user interface 2-125 for controlling theoperation of the instrument. The user interface 2-125 is configured toallow a user to input information into the instrument, such as commandsand/or settings used to control the functioning of the instrument. Insome embodiments, the user interface 2-125 may include buttons,switches, dials, and a microphone for voice commands. Additionally, theuser interface 2-125 may allow a user to receive feedback on theperformance of the instrument and/or assay chip, such as properalignment and/or information obtained by readout signals from thesensors on the sensor chip. In some embodiments, the user interface2-125 may provide feedback using a speaker to provide audible feedback,and indicator lights and/or a display screen for providing visualfeedback. In some embodiments, the instrument 2-120 includes a computerinterface 2-124 used to connect with a computing device 2-130. Anysuitable computer interface 2-124 and computing device 2-130 may beused. For example, the computer interface 2-124 may be a USB interfaceor a firewire interface. The computing device 2-130 may be any generalpurpose computer, such as a laptop, desktop, or tablet computer, or amobile device such as a cellular telephone. The computer interface 2-124facilitates communication of information between the instrument 2-120and the computing device 2-130. Input information for controlling and/orconfiguring the instrument 2-120 may be provided through the computingdevice 2-130 connected to the computer interface 2-124 of theinstrument. Additionally, output information may be received by thecomputing device 2-130 through the computer interface 2-124. Such outputinformation may include feedback about performance of the instrument2-120 and information from the readout signals of the sensor 2-122. Theinstrument 2-120 may also include a processing device 2-123 foranalyzing data received from the sensor 2-122. In some embodiments, theprocessing device 2-123 may be a general purpose processor (e.g., acentral processing unit (CPU), a field programmable gate array (FPGA) ora custom integrated circuit, such as an application specific integratedcircuit (ASIC). In some embodiments, the processing of data from thesensor 1-122 may be performed by both the processing device 2-123 andthe external computing device 2-130. In other embodiments, the computingdevice 2-130 may be omitted and processing of data from the sensor 2-122may be performed solely by processing device 2-123.

When the excitation source 2-121 illuminates the assay chip 2-110 withexcitation energy, samples within one or more pixels of the assay chip2-110 may be excited. In some embodiments, a specimen is labeled withmultiple markers and the multiple markers, each associated with adifferent sample within the specimen, are identifiable by the emissionenergy. The path from the sample well 2-111 to the sensor 2-122 mayinclude one or more components that aid in identifying the multiplemarkers based on emission energy. Components may focus emission energytowards the sensor 2-122 and may additionally or alternatively spatiallyseparate emission energies that have different characteristic energies,and therefore different wavelengths. In some embodiments, the assay chip2-110 may include components that direct emission energy towards thesensor 2-122 and the instrument 2-120 may include components forspatially separating emission energy of different wavelengths. Forexample, optical filters or diffractive optics may be used to couple thewavelength of the emission energy to a spatial degree of freedom. Thesensor or sensor region may contain multiple sub-sensors configured todetect a spatial distribution of the emission energy that depends on theradiation pattern. Luminescent tags that emit different emissionenergies and/or spectral ranges may form different radiation patterns.The sensor or sensor region may detect information about the spatialdistribution of the emission energy that can be used to identify amarker among the multiple markers.

The emission energy from the sample in the sample well 2-110 may bedetected by the sensor 2-122 and converted to at least one electricalsignal. The electrical signals may be transmitted along conducting linesin the circuitry of the instrument 2-120 and processed and/or analyzedby the processing device 2-123 and/or computing device 2-130.

FIG. 2-2 is a top view of the assay chip 2-110 and the top view of thesensor chip 2-260 and illustrates the correspondence between the pixelsof the two chips. The assay chip 2-110 comprises a plurality of pixels,each pixel including a sample well 2-111 formed in a conductive material2-221. The sensor chip 2-260 also comprises a plurality of pixels, eachpixel including a sensor 2-121 formed in or on a substrate 2-247. Thearrows in FIG. 2-2 illustrate the correspondence between two of thepixels of the assay chip 2-110 and two of the pixels of the sensor chip2-260. While not illustrated for the sake of clarity, each pixel of theassay chip 2-110 is associated with a pixel of the sensor chip 2-260.

An overview of some components associated with a single pixel of theassay chip 2-110 and a single pixel of the sensor chip 2-260 isillustrated in FIG. 2-3. The apparatus 2-100 comprises both the assaychip 2-110 and the instrument 2-120. In some embodiments, the assay chip2-110 is a disposable chip designed for the analysis of a singlespecimen. The assay chip 2-110 includes one or more metal layers 2-221,one or more dielectric layers 2-225 and focusing elements 2-227. In someembodiments, metal layer 2-221 includes a stack of layers, some of whichmay include absorbing layers. The instrument 2-120 includes one or moreexcitation sources 2-250, at least one polychroic mirror 2-230, and thesensor chip 2-260, which may include filtering elements 2-241, spectralsorting elements 2-243, focusing elements 2-245 and at least one sensor2-122 in or on the substrate 2-247. While FIG. 2-3 illustrates only asingle pixel of the assay chip 2-110 and only a single pixel of thesensor chip 2-260, some components of the instrument 2-120, such as theexcitation source 2-250, the polychroic mirror 2-230 and filteringelements 2-241, may be common to a plurality of the pixels. For example,in some embodiments, a single excitation source 2-250 and polychroicmirror 2-230 may direct the excitation energy to every pixel of theassay chip 2-110.

In some embodiments, the specimen may include bodily fluid, such asblood, urine or saliva. The sample well 2-211 within the metal layer2-221 forms a sample volume for a sample from the specimen to enter. Theopenings at the end of the sample well 2-211 may be referred to as ananoaperture. The nanoaperture may have a width that is smaller than thewavelength of the excitation energy 2-251 emitted by excitation source2-250. A portion of the specimen, referred to as a sample, may enter thesample volume defined by the sample well 2-211. The sample may be anyparticle, molecule, protein, genetic material or any other samplepresent in the specimen.

Excitation source 2-250 emits the excitation energy 2-251, which isdirected towards the sample well 2-211 to illuminate the sample. In someembodiments, the excitation source 2-251 may be a single light sourcethat provides excitation energy for all the pixel of the assay chip2-110. The polychroic mirror 2-230 reflects light from the excitationsource 2-250 and directs the excitation energy 2-251 towards one or moresample wells 2-211 of the assay chip 2-110. Thus, in some embodiments,there may be only a single polychroic mirror 2-230 that directs theexcitation energy towards all the sample wells, rather than each pixelbeing associated with its own polychroic mirror. Similarly, there may bea one-to many relationship between other optical elements used to directthe excitation energy towards the sample wells 2-211.

A concentric circular grating 2-223 may be formed adjacent to the bottomnanoaperture of the sample well 2-211. The concentric circular gratings2-223 may protrude from a bottom surface of the metal layer 2-221. Thesample well 2-211 may be located at or near the center of the circulargrating 2-223. Both the sub-wavelength scale of the nanoaperture of thesample well 2-211 and the concentric circular gratings 2-223 create afield enhancement effect that increases the intensity of the excitationenergy in the sample well 2-211, resulting in increased coupling of theexcitation energy to a sample present in the sample well 2-211. At leastsome of the time, the sample absorbs a photon from the excitation energyand emits a photon (referred to as “emission energy” 2-253) with anenergy less than that of the excitation energy 2-251. The emissionenergy 2-253 may be emitted in a downward direction. The circulargratings 2-223 act as plasmonic elements which may be used to decreasethe spread of the emission energy 2-253 and direct the emission energy2-253 towards an associated sensor.

The emission energy 2-253 travels through the dielectric layer 2-225,which may be a spacer layer used to allow the emission energy 2-253 topropagate some distance. The dielectric layer 2-225 may also providestructural strength to the assay chip 2-110. The emission energy 2-253then travels through one or more focusing elements 2-227 used to furtherdirect the emission energy 2-253 to the sensor 2-122 in the associatedpixel of the sensor chip 2-2260 within the instrument 2-120.

The polychroic mirror 2-230 then transmits the emission energy 2-253 andreflects a portion of any excitation energy 2-251 reflected from theassay chip 2-110. The portion of the excitation light that is notreflected by the assay chip 2-110 is either transmitted through theassay chip or absorbed by the assay chip. To further reduce the amountof excitation energy 2-251 reflected by the assay chip 2-110 and notreflected by the polychroic mirror 2-230, filtering elements 2-241 maybe disposed in the optical path towards the sensor chip 2-260. Thefiltering elements 2-241 may include, by way of example and notlimitation, a broadband filter, a notch filter or an edge filter, whichtransmit emission energy 2-253 but absorb and/or reflect excitationenergy 2-251.

In embodiments, to facilitate using spectral properties of the emissionenergy 2-253 to determine the identity of the marker in the sample well2-211, spectral sorting elements 2-243 may be included on the sensorchip 2-260 to couple the spectral degree of freedom of the emissionenergy 2-253 to the direction the emission energy 2-253 is traveling.For example, a diffractive optical element may be used to directemission energy 2-253 of a first wavelength in a first direction andemission energy 2-253 of a second wavelength in a second direction. Oneor more focusing elements 2-245 may be used to direct the spectrallysorted light onto the sensor 2-122. The sensor 2-122 may include one ormore sub-sensors (not shown), each of which is associated with adifferent wavelength of the emission energy 2-253 based on theredirection of light of different wavelengths by the spectral sortingelement 2-243.

The above description of FIG. 2-3 is an overview of some, but notnecessarily all, of the components of the apparatus according to someembodiments. In some embodiments, one or more elements of FIG. 2-3 maybe absent or in a different location. The components of the assay chip2-210 and instrument 2-220 are described in more detail below.

The assay chip 2-110 and the instrument 2-120 may be mechanicallyaligned, detachably coupled and separable from one another. Theinstrument 2-120 may include an instrument housing, inside which amounting board 2-405 is disposed. FIG. 2-4 illustrates at least some ofthe components that may be included on the mounting board 2-405 of theinstrument 2-120. The mounting board 2-405, which may include a printedcircuit board, may have the sensor chip 2-260 (not visible in FIG. 2-4),a heat sink 2-407 and an optical housing 2-401 mounted to it. Thevarious optical components of the instrument 2-120 may be disposedwithin the optical housing 2-401. In some embodiments, instrumenthousing and mounting board may be any suitable size. For example, themounting board may be substantially circular with a diameter of 7-8″.

The assay chip 2-110 couples to the optical housing 2-401 to ensurealignment with the optical components within the optical housing 2-401.A chip holder frame 3-102 may be aligned with an opening of the opticalhousing 2-401. Preferably, the assay chip 2-110 may be detachablycoupled to the instrument 2-120. For example, magnetic components 2-403a through 2-403 b of any suitable shape, such as magnetic cylinders, maybe placed around an opening of the optical housing 3-401 through whichexcitation energy exits the optical housing 2-401. Additionally, themagnetic components 2-403 a through 2-403 c may be calibrated such thatthe chip holder frame 3-102 is held in alignment with the opening. Thechip holder frame may be positioned with a micron-level accuracy usingthe alignment cylinders. In some embodiments, three magnetic cylinders2-403 a through 2-403 b are used to create chip holder frame alignment.However, embodiments are not so limited and any suitable number ofmagnetic, spring-loaded, pneumatic or other such components may be usedto hold the chip in place in an aligned configuration. For example, thechip holder frame 3-102 may be held in place with a non-magneticelement, such as a spring, air pressure, or suction from a vacuum.Optionally, the chip holder frame 3-102 may be constructed using anystiff material suitable for positioning the chip in alignment with theoptical block.

According to some aspects of the present application, when the chip isconnected to the system, the distance between the sample wells and thesensors can be kept small. In some embodiments, the optical distancebetween the sample wells and the sensors may be less than 30 cm, lessthan 10 cm, less than 5 cm, or less than 1 cm.

III. Assay Chip

In some embodiments, the assay chip 2-110 does not include any activeelectronic components. Both the excitation source 2-250 and the sensor2-122 for each pixel are located off-chip in the instrument 2-120.

In some embodiments, the assay chip 2-110 may be housed in a chip holderframe 3-102 as illustrated in FIG. 3-1A. The chip holder frame 3-102 maybe disposable and may be disposed of along with the assay chip 2-110after a single use. The assay chip 2-110 may be situated on theunderside of the chip holder frame 3-102, as illustrated in FIG. 3-1B.The chip holder frame 3-102 may comprise any suitable ferromagneticmetal, such as steel, such that the magnetic components 2-403 a through2-403 b fixed to the optical housing 2-401 hold the chip holder frame3-102, and thus the assay chip 2-110, in place. In some embodiments, thechip holder frame 3-102 may be attached to the top surface of theoptical housing 2-401 as illustrated in FIG. 2-4.

In other embodiments, illustrated in FIG. 3-1C, the assay chip may beattached to a top surface of the chip holder frame 3-102. A plastic cap3-103 surrounds the assay chip 2-110 such that the pixel array of theassay chip 2-110 is exposed via an opening in the plastic cap 3-103. Auser of the assay chip 2-110 may place a specimen into the opening ofthe plastic cap 3-103. By being in contact with the top surface of theassay chip 2-110, the samples within the specimen may be introduced toone or more of the plurality of pixels of the assay chip 2-110 foranalysis. In some embodiments, no fluidic channels or device fordelivering portions of the sample to the pixels via forced fluid floware necessary.

A. Sample Well Layer

As illustrated in FIG. 2-3, and in more detail at FIG. 3-2, someembodiments include a sample well 2-211 formed at one or more pixels ofthe assay chip 2-110. A sample well may comprise a small volume orregion formed within metal layer 2-221 and arranged such that samplesmay diffuse into and out of the sample well from a specimen deposited onthe surface of the assay chip 2-110. In various embodiments, a samplewell 2-211 may be arranged to receive excitation energy from anexcitation source 2-250. Samples that diffuse into the sample well maybe retained, temporarily or permanently, within an excitation region3-215 of the sample well by an adherent 3-211. In the excitation region,a sample may be excited by excitation energy (e.g., excitation light3-245), and subsequently emit energy that may be observed and evaluatedto characterize the sample.

In further detail of operation, at least one sample 3-101 to be analyzedmay be introduced into a sample well 2-211, e.g., from a specimen (notshown) containing a fluid suspension of samples. Excitation energy 3-245from an excitation source 2-250 in the instrument 2-120 may excite thesample or at least one tag (also referred to as a biological marker,reporter, or probe) attached to the sample or otherwise associated withthe sample while it is within an excitation region 3-215 within thesample well. According to some embodiments, a tag may be a luminescentmolecule (e.g., a luminescent tag or probe) or quantum dot. In someimplementations, there may be more than one tag that is used to analyzea sample (e.g., distinct tags that are used for single-molecule geneticsequencing as described in “Real-Time DNA Sequencing from SinglePolymerase Molecules,” by J. Eid, et al., Science 323, p. 133 (2009),which is incorporated by reference). During and/or after excitation, thesample or tag may emit emission energy. When multiple tags are used,they may emit at different characteristic energies (and therefore havedifferent wavelengths) and/or emit with different temporalcharacteristics. The emissions from the sample well 2-211 may radiate toa sensor 2-122 on the instrument 2-120 where they are detected andconverted into electrical signals that can be used to characterize thesample.

According to some embodiments, a sample well 2-211 may be a partiallyenclosed structure, as depicted in FIG. 3-2. In some implementations, asample well 2-211 comprises a sub-micron-sized hole or opening(characterized by at least one transverse dimension D_(sw)) formed in atleast one layer of material 2-221. The transverse dimension of thesample well may be between approximately 20 nanometers and approximately1 micron, according to some embodiments, though larger and smaller sizesmay be used in some implementations. A volume of the sample well 2-211may be between about 10⁻²¹ liters and about 10⁻¹⁵ liters, in someimplementations. A sample well may be formed as a waveguide that may, ormay not, support a propagating mode. In some embodiments, a sample wellmay be formed as a zero-mode waveguide (ZMW) having a cylindrical shape(or similar shape) with a diameter (or largest transverse dimension)D_(sw). A ZMW may be formed in a single metal layer as a nano-scale holethat does not support a propagating optical mode through the hole.

Because the sample well 2-211 has a small volume, detection ofsingle-sample events (e.g., single-molecule events) at each pixel may bepossible even though samples may be concentrated in an examined specimenat concentrations that are similar to those found in naturalenvironments. For example, micromolar concentrations of the sample maybe present in a specimen that is placed in contact with the assay chip,but at the pixel level only about. Sample wells of the assay 2-110 aresized such that, statistically, they most likely contain no sample orone sample, so that single molecule analysis may be performed. Forexample, in some embodiments 30-40% of the sample wells contain a singlesample. However, sample wells may contain more than one sample. Becausesingle-molecule or single-sample events may be analyzed at each pixel,the assay chip makes it possible to detect rare events that mayotherwise go unnoticed in ensemble averaged measurements.

A transverse dimension D_(sw) of a sample well may be between about 500nanometers (nm) and about one micron in some embodiments, between about250 nm and about 500 nm in some embodiments, between about 100 nm andabout 250 nm in some embodiments, and yet between about 20 nm and about100 nm in some embodiments. According to some implementations, atransverse dimension of a sample well is between approximately 80 nm andapproximately 180 nm, or between approximately one-quarter andone-eighth of the excitation wavelength or emission wavelength.According to other implementations, a transverse dimension of a samplewell is between approximately 120 nm and approximately 170 nm. In someembodiments, the depth or height of the sample well 2-211 may be betweenabout 50 nm and about 500 nm. In some implementations, the depth orheight of the sample well 2-211 may be between about 80 nm and about 200nm.

A sample well 2-211 having a sub-wavelength, transverse dimension canimprove operation of a pixel 2-100 of an assay chip 2-110 in at leasttwo ways. For example, excitation energy 3-245 incident on the samplewell from a side opposite the specimen may couple into the excitationregion 3-215 with exponentially decreasing power, and not propagatethrough the sample well to the specimen. As a result, excitation energyis increased in the excitation region where it excites a sample ofinterest, and is reduced in the specimen where it could excite othersamples that would contribute to background noise. Also, emission from asample retained at a base of the well is preferably directed toward thesensor on the instrument 2-120, since emission cannot propagate upthrough the sample well. Both of these effects can improvesignal-to-noise ratio at the pixel. The inventors have recognizedseveral aspects of the sample well that can be improved to further boostsignal-to-noise levels at the pixel. These aspects relate to well shapeand structure, and placement relative to adjacent optical and plasmonicstructures (described below) that aid in coupling excitation energy tothe sample well and emitted energy from the sample well.

According to some embodiments, a sample well 2-211 may be formed as asub-cutoff nano-aperture (SCN), which does not support a propagatingmode. For example, the sample well 2-211 may comprise acylindrically-shaped hole or bore in a conductive layer 2-221. Thecross-section of a sample well need not be round, and may be elliptical,square, rectangular, or polygonal in some embodiments. Excitation energy3-245 (e.g., visible or near infrared radiation) may enter the samplewell through an entrance aperture 3-212 that may be defined by walls3-214 of the sample well 2-211 at a first end of the well, as depictedin FIG. 3-2. When formed as an SCN, the excitation energy 3-245 maydecay exponentially along the SCN. In some implementations, thewaveguide may comprise an SCN for emitted energy from the sample, butmay not be an SCN for excitation energy. For example, the aperture andwaveguide formed by the sample well may be large enough to support apropagating mode for the excitation energy, since it may have a shorterwavelength than the emitted energy. The emission, at a longerwavelength, may be beyond a cut-off wavelength for a propagating mode inthe waveguide. According to some embodiments, the sample well 2-211 maycomprise an SCN for the excitation energy 3-245, such that the greatestintensity of excitation energy is localized to an excitation region3-215 of the sample well at an entrance to the sample well 2-211 (e.g.,localized near the interface between layer 3-235 and layer 2-221 asdepicted in FIG. 3-2). Such localization of the excitation energy canincrease the emission energy density from the sample, and furtherconfine the excitation energy near the entrance aperture 3-212, therebylimiting the observed emission to a single sample (e.g., a singlemolecule).

An example of excitation localization near an entrance of a sample wellthat comprises an SCN is depicted in FIG. 3-3. A numerical simulationwas carried out to determine intensity of excitation energy within andnear a sample well 2-211 formed as an SCN. The results show that theintensity of the excitation energy is about 70% of the incident energyat an entrance aperture of the sample well and drops to about 20% of theincident intensity within about 100 nm in the sample well. For thissimulation, the characteristic wavelength of the excitation energy was633 nm and the diameter of the sample well 2-211 was 140 nm. The samplewell 2-211 was formed in a layer of gold metal. Each horizontal divisionin the graph is 50 nm. As shown by the graph, more than one-half of theexcitation energy received in the sample well is localized to about 50nm within the entrance aperture 3-212 of the sample well 2-211.

To improve the intensity of excitation energy that is localized at thesample well 2-211, other sample well structures were developed andstudied by the inventors. FIG. 3-4 depicts an embodiment of a samplewell that includes a cavity or divot 3-216 at an excitation end of thesample well 2-211. As can be seen in the simulation results of FIG. 3-3,a region of higher excitation intensity exists just before the entranceaperture 2-212 of the sample well. Adding a divot 3-216 to the samplewell 2-211 allows a sample to move into a region of higher excitationintensity, according to some embodiments. In some implementations, theshape and structure of the divot alters the local excitation field(e.g., because of a difference in refractive index between the layer3-235 and fluid of the specimen in the sample well), and can furtherincrease the intensity of the excitation energy in the divot.

The divot may have any suitable shape. The divot may have a transverseshape that is substantially equivalent to a transverse shape of thesample well, e.g., round, elliptical, square, rectangular, polygonal,etc. In some embodiments, the sidewalls of the divot may besubstantially straight and vertical, like the walls of the sample well.In some implementations, the sidewalls of the divot may be sloped and/orcurved, as depicted in the drawing. The transverse dimension of thedivot may be approximately the same size as the transverse dimension ofthe sample well in some embodiments, may be smaller than the transversedimension of the sample well in some embodiments, or may be larger thanthe transverse dimension of the sample well in some embodiments. Thedivot 3-216 may extend between approximately 10 nm and approximately 200nm beyond the metallic layer 2-221 of the sample well. In someimplementations, the divot may extend between approximately 50 nm andapproximately 150 nm beyond the metallic layer 2-221 of the sample well.By forming the divot, the excitation region 3-215 may extend outside themetallic layer 2-221 of the sample well, as depicted in FIG. 3-4.

FIG. 3-5 depicts improvement of excitation energy at the excitationregion for a sample well containing a divot (shown in the leftsimulation image). For comparison, the excitation field is alsosimulated for a sample well without a divot, shown on the right. Thefield magnitude has been converted from a color rendering in theseplots, and the dark region at the base of the divot represents higherintensity than the light region within the sample well. The dark regionsabove the sample well represents the lowest intensity. As can be seen,the divot allows a sample 3-101 to move to a region of higher excitationintensity, and the divot also increases the localization of region ofhighest intensity at an excitation end of the sample well. Note that theregion of high intensity is more distributed for the sample well withoutthe divot. In some embodiments, the divot 3-216 provides an increase inexcitation energy at the excitation region by a factor of two or more.In some implementations, an increase of more than a factor of two can beobtained depending on the shape and depth of the divot. In thesesimulations, the sample well comprises a layer of Al that is 100 nmthick, with a divot that is 50 nm deep, with excitation energy at 635 nmwavelength.

FIG. 3-6 depicts another embodiment of a sample well 2-211 in which thesample well, including the divot, are formed over a protrusion 3-615 ata surface of a substrate. A resulting structure for the sample well mayincrease the excitation energy at the sample by more than a factor oftwo compared to a sample well shown in FIG. 3-2, and may direct emissionfrom the sample well toward the sensor in the instrument 2-120.According to some embodiments, a protrusion 3-615 is patterned in afirst layer 3-610 of material. The protrusion may be formed as acircular pedestal or a ridge with rectangular cross-section in someimplementations, and a second layer 3-620 of material may be depositedover the first layer and the protrusion. At the protrusion, the secondlayer may form a shape above the protrusion that approximates acylindrical portion 3-625, as depicted. In some embodiments, aconductive layer 3-230 (e.g., a reflective metal) may be deposited overthe second layer 3-620 and patterned to form a sample well 3-210 in theconductive layer above the protrusion. A divot 3-216 may then be etchedinto the second layer. The divot may extend between about 50 nm andabout 150 nm below the conductive layer 3-230. According to someembodiments, the first layer 3-610 and second layer 3-620 may beoptically transparent, and may or may not be formed of a same material.In some implementations, the first layer 3-610 may be formed from anoxide (e.g., SiO2) or a nitride (e.g., Si3N4), and the second layer3-620 may be formed from an oxide or a nitride.

According to some embodiments, the conductive layer 3-230 above theprotrusion 3-625 is shaped approximately as a spherical reflector 3-630.The shape of the spherical portion may be controlled by selection of theprotrusion height h, diameter or transverse dimension w of theprotrusion, and a thickness t of the second layer 3-620. The location ofthe excitation region and position of the sample can be adjusted withrespect to an optical focal point of the cylindrical reflector byselection of the divot depth d. It may be appreciated that the sphericalreflector 3-630 can concentrate excitation energy at the excitationregion 3-215, and can also collect energy emitted from a sample andreflect and concentrate the radiation toward the sensor 3-260.

As noted above, a sample well may be formed in any suitable shape, andis not limited to only cylindrical shapes. In some implementations, asample well may be conic, tetrahedron, pentahedron, etc. FIG. 3-7Athrough FIG. 3-7F illustrates some example sample well shapes andstructures that may be used in some embodiments. A sample well 2-211 maybe formed to have an first aperture 2-212 that is larger than a secondaperture 2-218 for the excitation energy, according to some embodiments.The sidewalls of the sample well may be tapered or curved. Forming asample well in this manner can admit more excitation energy to theexcitation region, yet still appreciably attenuate excitation energythat travels toward the specimen. Additionally, emission radiated by asample may preferentially radiate toward the end of the sample well withthe larger aperture, because of favorable energy transfer in thatdirection.

In some embodiments, a divot 3-216 may have a smaller transversedimension than the base of the sample well, as depicted in FIG. 3-7B. Asmaller divot may be formed by coating sidewalls of the sample well witha sacrificial layer before etching the divot, and subsequently removingthe sacrificial layer. A smaller divot may be formed to retain a samplein a region that is more equidistant from the conductive walls of thesample well. Retaining a sample equidistant from the walls of the samplewell may reduce undesirable effects of the sample well walls on theradiating sample, e.g., quenching of emission and/or altering ofradiation lifetimes.

FIG. 3-7C and 3-7D depict another embodiment of a sample well. Accordingto this embodiment, a sample well 2-211 may compriseexcitation-energy-enhancing structures 3-711 and an adherent 3-211formed adjacent the excitation-energy-enhancing structures. Theenergy-enhancing structures 3-711 may comprise surface plasmon ornano-antenna structures formed in conductive materials on an opticallytransparent layer 3-235, according to some embodiments. FIG. 3-7Cdepicts an elevation view of the sample well 2-211 and nearby structure,and FIG. 3-7D depicts a plan view. The excitation-energy-enhancingstructures 3-711 may be shaped and arranged to enhance excitation energyin a small localized region. For example, the structures may includepointed conductors having acute angles at the sample well that increasethe intensity of the excitation energy within an excitation region3-215. In the depicted example, the excitation-energy-enhancingstructures 3-711 are in the form of a bow-tie. Samples 3-101 diffusinginto the region may be retained, temporarily or permanently, by theadherent 3-211 and excited by excitation energy that may be deliveredfrom an excitation source 2-250 located in the instrument 2-120.According to some embodiments, the excitation energy may drivesurface-plasmon currents in the energy-enhancing structures 3-711. Theresulting surface-plasmon currents may produce high electric fields atthe sharp points of the structures 3-711, and these high fields mayexcite a sample retained in the excitation region 3-215. In someembodiments, a sample well 2-211 depicted in FIG. 3-7C may include adivot 3-216.

Another embodiment of a sample well is depicted in FIG. 3-7E, and showsan excitation-energy-enhancing structure 3-720 formed along interiorwalls of the sample well 2-211. The excitation-energy-enhancingstructure 3-720 may comprise a metal or conductor, and may be formedusing an angled (or shadow), directional deposition where the substrateon which the sample well is formed is rotated during the deposition.During the deposition, the base of the sample well 2-211 is obscured bythe upper walls of the well, so that the deposited material does notaccumulate at the base. The resulting structure 3-720 may form an acuteangle 3-722 at the bottom of the structure, and this acute angle of theconductor can enhance excitation energy within the sample well.

In an embodiment as depicted in FIG. 3-7E, the material 3-232 in whichthe sample well is formed need not be a conductor, and may be anysuitable material such as a dielectric material. According to someimplementations, the sample well 2-211 and excitation-energy-enhancingstructure 3-720 may be formed at a blind hole etched into a dielectriclayer 3-235, and a separate layer 3-232 need not be deposited.

In some implementations, a shadow evaporation may be subsequentlyperformed on the structure shown in FIG. 3-7E to deposit a metallic orconductive energy-enhancing structure, e.g., a trapezoidal structure orpointed cone at the base of the sample well, as depicted by the dashedline. The energy-enhancing structure may enhance the excitation energywithin the well via surface plasmons. After the shadow evaporation, aplanarizing process (e.g., a chemical-mechanical polishing step or aplasma etching process) may be performed to remove or etch back thedeposited material at the top of the sample well, while leaving theenergy-enhancing structure within the well.

In some embodiments, a sample well 2-211 may be formed from more than asingle metal layer. FIG. 3-7F illustrates a sample well formed in amulti-layer structure, where different materials may be used for thedifferent layers. According to some embodiments, a sample well 2-211 maybe formed in a first layer 3-232 (which may be a semiconducting orconducting material), a second layer 3-234 (which may be an insulator ordielectric), and a third layer 2-221 (which may be a conductor orsemiconductor). In some embodiments, a degeneratively-dopedsemiconductor or graphene may be used for a layer of the sample well. Insome implementations, a sample well may be formed in two layers, and inother implementations a sample well may be formed in four or morelayers. In some embodiments, multi-layer materials used for forming asample well may be selected to increase surface-plasmon generation at abase of the sample well or suppress surface-plasmon radiation at a topof the well. In some embodiments, multi-layer materials used for forminga sample well may be selected to suppress excitation energy frompropagating beyond the sample well and multi-layer structure into thebulk specimen.

In some embodiments, multi-layer materials used for forming a samplewell may be selected to increase or suppress interfacial excitons whichmay be generated by excitation energy incident on the sample well. Forexample, multi-excitons, such as biexcitons and triexitons, may begenerated at an interface between two different semiconductor layersadjacent a sample well. The sample well may be formed in both the metallayer and the first semiconductor layer such that the interface betweenthe first semiconductor layer and a second semiconductor layer is at anexcitation region 3-215 of the sample well. Interfacial excitons mayhave longer lifetimes than excitons within the volume of a singlesemiconductor layer, increasing the likelihood that the excitons willexcite a sample or tag via FRET or DET. In some embodiments, at leastone quantum dot at which multi-excitons may be excited may be attachedto a bottom of the sample well (e.g., by a linking molecule). Excitonsexcited at a quantum dot may also have longer lifetimes than excitonswithin the volume of a single semiconductor layer. Interfacial excitonsor excitons generated at a quantum dot may increase the rate of FRET orDET, according to some embodiments.

Various materials may be used to form sample wells described in theforegoing embodiments. According to some embodiments, a sample well2-211 may be formed from at least one layer of material 2-221, which maycomprise any one of or a combination of a conductive material, asemiconductor, and an insulator. In some embodiments, the sample well2-211 comprises a highly conductive metallic layer, e.g., gold, silver,aluminum, copper. In some embodiments, the layer 2-221 may comprise amulti-layer stack that includes any one of or a combination of gold,silver, aluminum, copper, titanium, titanium nitride, and chromium. Insome implementations, other metals may be used additionally oralternatively. According to some embodiments, a sample well may comprisean alloy such as AlCu or AlSi.

In some embodiments, the multiple layers of different metals or alloysmay be used to form a sample well. In some implementations, the materialin which the sample well 2-211 is formed may comprise alternating layersof metals and non-metals, e.g., alternating layers of metal and one ormore dielectrics. In some embodiments, the non-metal may include apolymer, such as polyvinyl phosphonic acid or a polyethylene glycol(PEG)-thiol.

A layer 2-221 in which a sample well is formed may be deposited on oradjacent to at least one optically transparent layer 3-235, according tosome embodiments, so that excitation energy (e.g., in the form ofvisible or near-infrared radiation) and emission energy (e.g., in theform of visible or near-infrared radiation) may travel to and from thesample well 2-211 without significant attenuation. For example,excitation energy from an excitation source 2-250 may pass through theat least one optically transparent layer 2-235 to the excitation region3-215, and emission from the sample may pass through the same layer orlayers to the sensor 2-250.

In some embodiments, at least one surface of the sample well 2-211 maybe coated with one or more layers 3-211, 3-280 of material that affectthe action of a sample within the sample well, as depicted in FIG. 3-8.For example, a thin dielectric layer 3-280 (e.g., alumina, titaniumnitride, or silica) may be deposited as a passivating coating onsidewalls of the sample well. Such a coating may be implemented toreduce sample adhesion of a sample outside the excitation region 3-215,or to reduce interaction between a sample and the material 2-221 inwhich the sample well 2-211 is formed. The thickness of a passivatingcoating within the sample well may be between about 5 nm and about 50nm, according to some embodiments.

In some implementations, a material for a coating layer 3-280 may beselected based upon an affinity of a chemical agent for the material, sothat the layer 3-280 may be treated with a chemical or biologicalsubstance to further inhibit adhesion of a sample species to the layer.For example, a coating layer 3-280 may comprise alumina, which may bepassivated with a polyphosphonate passivation layer, according to someembodiments. Additional or alternative coatings and passivating agentsmay be used in some embodiments.

According to some embodiments, at least a bottom surface of the samplewell 2-211 and/or divot 3-216 may be treated with a chemical orbiological adherent 3-211 (e.g., biotin) to promote retention of asample. The sample may be retained permanently or temporarily, e.g., forat least a period of time between about 0.5 milliseconds and about 50milliseconds. In another embodiment, the adherent may promote temporaryretention of a sample 3-101 for longer periods. Any suitable adherentmay be used in various embodiments, and is not limited to biotin.

According to some embodiments, the layer of material 3-235 adjacent thesample well may be selected based upon an affinity of an adherent forthe material of that layer. In some embodiments, passivation of thesample well's side walls may inhibit coating of an adherent on thesidewalls, so that the adherent 3-211 preferentially deposits at thebase of the sample well. In some embodiments, an adherent coating mayextend up a portion of the sample well's sidewalls. In someimplementations, an adherent may be deposited by an anisotropic physicaldeposition process (e.g., evaporation, sputtering), such that theadherent accumulates at the base of a sample well or divot and does notappreciably form on sidewalls of the sample well.

Various fabrication techniques may be employed to fabricate sample wells2-211 for an assay chip. A few example processes are described below,but the invention is not limited to only these examples.

The sample well 2-211 may be formed by any suitable micro- ornano-fabrication process, which may include, but is not limited to,processing steps associated with photolithography, deep-ultravioletphotolithography, immersion photolithography, near-field optical contactphotolithography, EUV lithography, x-ray lithography, nanoimprintlithography, interferometric lithography, step-and-flash lithography,direct-write electron beam lithography, ion beam lithography, ion beammilling, lift-off processing, reactive-ion etching, selective epitaxy,molecular self-assembly, organic synthesis, etc. According to someembodiments, a sample well 2-211 may be formed using photolithographyand lift-off processing. Example fabrication steps associated withlift-off processing of a sample well are depicted in FIG. 3-9. Althoughfabrication of only a single sample well or structure at a pixel istypically depicted in the drawings, it will be understood that a largenumber of sample wells or structures may be fabricated on a substrate(e.g., at each pixel) in parallel.

According to some embodiments, a layer 3-235 (e.g., an oxide layer) on asubstrate may be covered with an anti-reflection (ARC) layer 3-910 andphotoresist 3-920, as depicted in FIG. 3-9A. The photoresist may beexposed and patterned using photolithography and development of theresist. The resist may be developed to remove exposed portions orunexposed portions (depending on the resist type), leaving a pillar3-922 that has a diameter approximately equal to a desired diameter forthe sample well, as depicted in FIG. 3-9B. The height of the pillar maybe greater than a desired depth of the sample well.

The pattern of the pillar 3-922 may be transferred to the ARC layer3-910 via anisotropic, reactive ion etching (RIE), for example as shownin FIG. 3-9C. The region may then be coated with at least one material2-221, e.g., a conductor or metal, that is desired to form the samplewell. A portion of the deposited material, or materials, forms a cap3-232 over the pillar 3-922, as depicted in FIG. 3-9D. The resist andARC may then be stripped from the substrate, using a selective removalprocess (e.g., using a chemical bath with or without agitation whichdissolves at least the resist and releases or “lifts off” the cap). Ifthe ARC remains, it may be stripped from the substrate using a selectiveetch, leaving the sample well 3-210 as shown in FIG. 3-9E. According tosome embodiments, the sidewalls 3-214 of the sample well may be slopeddue to the nature of the deposition of the at least one material 2-221.

As used herein, a “selective etch” means an etching process in which anetchant selectively etches one material that is desired to be removed oretched at a higher rate (e.g., at least twice the rate) than the etchantetches other materials which are not intended to be removed.

Because the resist and ARC are typically polymer based, they areconsidered soft materials which may not be suitable for forming samplewells having high aspect ratios (e.g., aspect ratios greater than about2:1 with respect to height-to-width). For sample wells having higheraspect ratios, a hard material may be included in the lift-off process.For example, before depositing the ARC and photoresist, a layer of ahard (e.g., an inorganic material) may be deposited. In someembodiments, a layer of titanium or silicon nitride may be deposited.The layer of hard material should exhibit preferential etching over thematerial, or materials, 2-221 in which the sample well is formed. Afterthe photoresist is patterned, a pattern of the pillar may be transferredinto the ARC and the underlying hard material 3-930 yielding a structureas depicted in FIG. 3-9F. The photoresist and ARC may be then stripped,the material(s) 2-221 deposited, and a lift-off step performed to formthe sample well.

According to some embodiments, a lift-off process may be used to form asample well comprising energy-enhancing structures 3-711, as depicted inFIG. 3-7C and FIG. 3-7D.

An alternative process for forming a sample well is depicted in FIG.3-10. In this process, the sample well may be directly etched into atleast one material 2-211. For example, at least one material 2-211 inwhich a sample well is to be formed may be deposited on a substrate3-325. The layer may be covered by an ARC layer 3-910 and a photoresist3-920, as illustrated in FIG. 3-10A. The photoresist may be patterned toform a hole having a diameter approximately equal to a desired diameterof the sample well, as depicted in FIG. 3-10B. The pattern of the holemay be transferred to the ARC and through the layer 3-230 using ananisotropic, reactive ion etch, as shown in FIG. 3-10C for example. Theresist and ARC may be stripped, yielding a sample well as depicted inFIG. 3-10D. According to some embodiments, the sidewalls of a samplewell formed by etching into the layer of material 3-230 may be morevertical than sidewalls resulting from a lift-off process.

In some embodiments, the photoresist and ARC may be used to pattern ahard mask (e.g., a silicon nitride or oxide layer, not shown) over thematerial 2-221. The patterned hole may then be transferred to the hardmask, which is then used to transfer the pattern into the layer ofmaterial 2-221. A hard mask may allow greater etching depths into thelayer of material 2-221, so as to form sample wells of higher aspectratio.

It will be appreciated that lift-off processes and direct etchingfabrication techniques described above may be used to form a sample wellwhen multiple layers of different materials are used to form a stack ofmaterial 2-211 in which the sample well is formed. An example stack isshown in FIG. 2-11. According to some embodiments, a stack of materialmay be used to form a sample well to improve coupling of excitationenergy to the excitation region of a sample well, or to reducetransmission or re-radiation of excitation energy into the bulkspecimen. For example, an absorbing layer 3-942 may be deposited over afirst layer 3-940. The first layer may comprise a metal or metal alloy,and the absorbing layer may comprise a material that inhibits surfaceplasmons, e.g., amorphous silicon, TaN, TiN, or Cr. In someimplementations, a surface layer 3-944 may also be deposited topassivate the surface surrounding the sample well (e.g., inhibitadhesion of molecules).

Formation of a sample well including a divot 3-216 may be done in anysuitable manner. In some embodiments, a divot may be formed by etchingfurther into an adjacent layer 3-235, and/or any intervening layer orlayers, adjacent the sample well. For example, after forming a samplewell in a layer of material 2-221, that layer 2-221 may be used as anetch mask for patterning a divot, as depicted in FIG. 3-12. For example,the substrate may be subjected to a selective, anisotropic reactive ionetch so that a divot 3-216 may be etched into adjacent layer 3-235. Forexample, in an embodiment where the material 2-221 is metallic and theadjacent layer 3-235 silicon oxide, a reactive-ion plasma etch having afeed gas comprising CHF3 or CF4 may be used to preferentially removeexposed silicon oxide below the sample well and form the divot 3-216. Asused herein, “silicon oxide” generally refers to SiOx and may includesilicon dioxide, for example.

In some embodiments, conditions within the plasma (e.g., bias to thesubstrate and pressure) during an etch may be controlled to determinethe etch profile of the divot 3-216. For example, at low pressure (e.g.,less than about 100 mTorr) and high DC bias (e.g., greater than about20V), the etching may be highly anisotropic and form substantiallystraight and vertical sidewalls of the divot, as depicted in thedrawing. At higher pressures and lower bias, the etching may be moreisotropic yielding tapered and/or curved sidewalls of the divot. In someimplementations, a wet etch may be used to form the divot, which may besubstantially isotropic and form an approximately spherical divot thatmay extend laterally under the material 2-221, up to or beyond thesidewalls of the sample well.

FIG. 3-13A through FIG. 3-13C depict process steps that may be used toform a divot 3-216 having a smaller transverse dimension than the samplewell 2-211 (for example, a divot like that depicted in FIG. 3-7B). Insome implementations, after forming a sample well, a conformalsacrificial layer 3-960 may be deposited over a region including thesample well. According to some embodiments, the sacrificial layer 3-960may be deposited by a vapor deposition process, e.g., chemical vapordeposition (CVD), plasma-enhanced CVD, or atomic layer deposition (ALD).The sacrificial layer may then be etched back using a first anisotropicetch that is selective to the sacrificial layer 3-960, removes the layerfrom horizontal surfaces, leaves side wall coatings 3-962 on walls ofthe sample well, as depicted in FIG. 3-13B. The etch back may beselective and stop on the material 2-221 and adjacent layer 3-235 insome embodiments, or may be a non-selective, timed etch in someembodiments.

A second anisotropic etch that is selective to the adjacent layer 3-235may be executed to etch a divot 3-216 into the adjacent layer asdepicted in FIG. 3-13C. The sacrificial side wall coatings 3-962 maythen optionally be removed by a selective wet or dry etch. The removalof the sidewall coatings open up the sample well to have a largertransverse dimension than the divot 3-216.

According to some embodiments, the sacrificial layer 3-960 may comprisethe same material as the adjacent layer 3-235. In such embodiments, thesecond etch may remove at least some of the side wall coating 3-962 asthe divot is etched into the adjacent layer 3-235. This etch back of theside wall coating can form tapered sidewalls of the divot in someembodiments.

In some implementations, the sacrificial layer 3-960 may be formed from,or include a layer of, a material that is used to passivate thesidewalls of the sample well (e.g., reduce adhesion of samples at thesidewalls of the sample well). At least some of the layer 3-960 may thenbe left on the walls of the sample well after formation of the divot.

According to some embodiments, the formation of the sidewall coatings3-962 occurs after the formation of the divot. In such an embodiment thelayer 3-960 coats the sidewalls of the divot. Such a process may be usedto passivate the sidewalls of the divot and localize the sample at thecenter of the divot.

Process steps associated with depositing an adherent 3-211 at a base ofa sample well 2-211, and a passivation layer 3-280 are depicted in FIG.3-14. According to some embodiments, a sample well may include a firstpassivation layer 3-280 on walls of the sample well. The firstpassivation layer may be formed, for example, as described above inconnection with FIG. 3-13B or FIG. 3-8. In some embodiments, a firstpassivation layer 3-280 may be formed by any suitable deposition processand etch back. In some embodiments, a first passivation layer may beformed by oxidizing the material 3-230 in which the sample well isformed. For example, the sample well may be formed of aluminum, whichmay be oxidized to create a coating of alumina on sidewalls of thesample well.

An adherent 3-980 or an adherent precursor (e.g., a material whichpreferentially binds an adherent) may be deposited on the substrateusing an anisotropic physical deposition process, e.g., an evaporativedeposition, as depicted in FIG. 3-14A. The adherent or adherentprecursor may form an adherent layer 3-211 at the base of the samplewell, as depicted in FIG. 3-14B, and may coat an upper surface of thematerial 2-221 in which the sample well is formed. A subsequent angled,directional deposition depicted in FIG. 3-14C (sometimes referred to asa shadow deposition or shadow evaporation process) may be used todeposit a second passivation layer 2-280 over an upper surface of thematerial 2-221 without covering the adherent layer 3-211. During theshadow deposition process, the substrate may be rotated around an axisnormal to the substrate, so that the second passivation layer 3-280deposits more uniformly around an upper rim of the sample well. Aresulting structure is depicted in FIG. 3-14D, according to someembodiments. As an alternative to depositing the second passivationlayer, a planarizing etch (e.g., a CMP step) may be used to removeadherent from an upper surface of the material 3-230.

According to some implementations, an adherent layer 3-211 may bedeposited centrally at the base of a tapered sample well, as depicted inFIG. 3-15. For example, an adherent, or adherent precursor, may bedirectionally deposited, as depicted in FIG. 3-14A, in a tapered samplewell, formed as described above. Walls of the sample well may bepassivated by an oxidation process before or after deposition of theadherent layer 3-211. Adherent or precursor remaining on a surface ofthe material 2-221 may be passivated as described in connection withFIG. 3-14D. In some embodiments, an adherent on an upper surface of thematerial 2-221 may be removed by a chemical-mechanical polishing step.By forming an adherent layer, or an adherent layer precursor, centrallyat the base of a sample well, deleterious effects on emission from asample (e.g., suppression or quenching of sample radiation from samplewalls, unfavorable radiation distribution from a sample because it isnot located centrally with respect to energy coupling structures formedaround a sample well, adverse effects on luminescent lifetime for asample) may be reduced.

In some embodiments, lift-off patterning, etching, and depositionprocesses used to form the sample well and divot may be compatible withCMOS processes that are used to form integrated CMOS circuits on asensor chip. Accordingly, sensor may be fabricated using conventionalCMOS facilities and fabrication techniques, though custom or specializedfabrication facilities may be used in some implementations.

Variations of the process steps described above may be used to formalternative embodiments of sample wells. For example, a tapered samplewell such as depicted in FIG. 3-7A or FIG. 3-7B may be formed using anangled deposition process depicted in FIG. 3-14C. For the sample well ofFIG. 3-7B, the angle of deposition may be changed during the depositionprocess. For such embodiments, a sample well having substantiallystraight and vertical sidewalls may first be formed, and then additionalmaterial 2-221 deposited by an angled deposition to taper the sidewallsof the sample well.

B. Coupling Excitation Energy to the Sample Well

As illustrated in FIG. 2-1 and FIG. 2-3, excitation energy 2-251 fromthe excitation source 2-250 is guided to the sample well 2-211 usingcomponents of the instrument 2-120 and components of the assay chip2-110. This section describes the components of the assay chip 2-110that may aid in the coupling of excitation energy 2-251 to the samplewell 2-211.

Coupling of energy from an excitation source to a sample well may beimproved or affected by forming excitation-coupling structures withinand/or adjacent a sample well. Excitation-coupling structures maycomprise micro- or nano-scale structures fabricated around a sample wellin some embodiments, or may comprise structures or particles formed at asample well in some embodiments. Excitation-coupling structures mayaffect radiative excitation of a sample in some implementations, and mayaffect non-radiative excitation of a sample in some implementations. Invarious embodiments, radiative excitation-coupling structures mayincrease an intensity of excitation energy within an excitation regionof a sample well. Non-radiative excitation-coupling structures mayimprove and/or alter non-radiative energy-transfer pathways from anexcitation source (which may be radiative or non-radiative) to a sample.

C. Radiative Excitation-Coupling Structures

There are a number of different types of radiative, excitation-couplingstructures that may be used to affect coupling of excitation energy froman excitation source to an excitation region within a sample well. Someradiative coupling structures may be formed of a conductor (e.g.,include a metal layer), and support surface plasmon oscillations thatlocally affect the excitation energy (e.g., locally alter anelectromagnetic field) near and/or within the sample well. In somecases, surface-plasmon structures may enhance the excitation energywithin an excitation region of the sample well by a factor of two ormore. Some radiative coupling structures may alter the phase and/oramplitude of an excitation field to enhance excitation energy within asample well. Various embodiments of radiative excitation-couplingstructures are described in this section.

FIG. 4-1A depicts just one example of a surface-plasmon structure 4-120that may be used to enhance coupling of excitation energy into a samplewell. The drawing depicts a plan view of a region around asurface-plasmon structure 4-120, and represents results of a numericalsimulation of electric field intensity around the structure. The drawingdepicts a surface-plasmon structure comprising three triangular featureshaving sharp apexes that are located in close proximity to a sample well(not shown). According to some embodiments, a surface-plasmon structuremay comprise a metal or conductor (e.g., a patterned thin film of anyone or combination of the following metals or metal alloys: Al, Au, Ag,Ti, TiN). A thickness of the film may be between approximately 10 nm andapproximately 100 nm in some embodiments, though other thicknesses maybe used in other embodiments. A surface-plasmon structure, in someembodiments, may include sharp features 4-110 located in close proximityto a sample well (e.g., within about 100 nm).

FIG. 4-1B depicts a cross-section, elevation view of the surface-plasmonstructure of FIG. 4-1A, taken at the dashed line. The simulation shows alocalized, high-intensity region 4-505 of the excitation energy adjacentan apex of a triangle of the surface-plasmon structure. For thissimulation, the surface-plasmon structure 4-120 was located on adielectric layer 4-135 (silicon dioxide). The surface-plasmon structuretaps energy from an evanescent field of the waveguide, and enhances theintensity at the sample well.

In some embodiments, enhancement of excitation energy by asurface-plasmon structure may be localized to an extent that a deepsample well 2-211 is not needed. For example, if a high-intensity region4-505 is formed having a diameter of approximately 100 nm with a peakintensity value greater than about 80% of the intensity outside theregion, then a deep sample well may not be needed. Only samples withinthe high-intensity region 4-505 would contribute appreciable emissionfor purposes of detection.

When an incident electromagnetic field interacts with a surface-plasmonstructure, surface-wave currents are generated in the structure. Theshape of the structure can affect the intensity and distribution ofthese surface-plasmons. These localized currents can interact with andsignificantly alter and intensify the incident electromagnetic field inthe immediate vicinity of the surface-plasmon structure, e.g., asdepicted by the high-intensity region 4-505 in FIG. 4-1B. In someembodiments, an emitter (e.g., a fluorescing tag) that emits energy neara surface-plasmon structure can have its emission altered by thestructure, so as to alter a far-field radiation pattern from theemitter.

Another embodiment of a surface-plasmon structure 4-122 is depicted inthe plan view of FIG. 4-1C. The illustrated bow-tie structure comprisestwo triangular metallic structures located adjacent a sample well 2-211.The structures may be patterned below a sample well, for example, and/oradjacent an excitation region of the sample well. There may be a gap4-127 between the sample well and sharp features 4-125 of thesurface-plasmon structure, in some implementations. The gap 4-127 may bebetween approximately 10 nm and approximately 200 nm, according to someembodiments. In some implementations, the gap 4-127 may be betweenapproximately 10 nm and approximately 100 nm. The sharp features 4-125may comprise a point or sharp bend in an edge of the surface-plasmonstructure, as depicted in the drawing. The sharp features may have anysuitable shape. In some embodiments a bend radius of a sharp feature4-125 may be less than approximately five wavelengths associated withthe incident excitation energy. In some embodiments a bend radius of asharp feature 4-125 may be less than approximately two wavelengthsassociated with the incident excitation energy. In some embodiments abend radius of a sharp feature 4-125 may be less than approximately fivewavelengths associated with a surface-plasmon wave that is excited bythe incident excitation energy. In some embodiments a bend radius of asharp feature 4-125 may be less than approximately two wavelengthsassociated with a surface-plasmon wave that is excited by the incidentexcitation energy.

According to some embodiments, surface-plasmon structures 4-122 may bepatterned within a sample well 2-211 as illustrated in the elevationview of FIG. 4-1D. In some embodiments, a surface-plasmon structurewithin a sample well may comprise one or more fingers (e.g., metallicfingers) patterned onto sidewalls of the sample well, as depicted in thedrawing. FIG. 4-1E depicts a plan view of the sample well 2-211 showingthe surface-plasmon structures 4-122 formed on sidewalls within thesample well. In some embodiments, the lower ends of thesesurface-plasmon structures 4-122 form sharp features or bends where theelectromagnetic field will be enhanced. The surface-plasmon structures4-122 may, or may not, extend to a base of the sample well.

In some embodiments, the surface-plasmon structures 4-122 may bearranged to affect the polarization of the excitation energy and/oremitted energy from the sample well. For example, a pattern as depictedin FIG. 4-1E may be used to affect a preferred orientation of linear orelliptical excitation polarization and/or a preferred orientation oflinear or elliptical polarization from an emitter within the samplewell.

Surface-plasmon structures may be patterned in shapes other than thosedepicted in FIG. 4-1A through FIG. 4-1E. For example, surface-plasmonstructures may be patterned as regular or periodic structures, asdepicted in FIG. 4-2A, according to some embodiments. For example, asurface-plasmon structure may be patterned is an array of protrudingfeatures 4-210 on a lower surface of a material 2-221 in which thesample well 2-211 is formed. Periodic surface-plasmon structures may beformed in a regular array, for example, a grating, a grid, a lattice, acircular grating, a spiral grating, an elliptical grating, or any othersuitable structure. There may be a substantially uniform spacing sbetween the protrusions 4-210 of a surface-plasmon structure. In someimplementations, the spacing s may have any value between approximately40 nm and approximately 250 nm. According to some embodiments, theprotrusions may have a height h between approximately 20 nm andapproximately 100 nm. In some implementations, the spacing s may benon-uniform or may be chirped (having a decreasing value at largerradial distances). In some embodiments, the protrusions 5-210 of asurface-plasmon structure may be patterned as a Fresnel zone plate.According to some embodiments, a surface-plasmon structure of 4-210 maybe formed adjacent to a transparent layer and/or dielectric layer 3-235.In some embodiments, the spacing between the protrusions 4-210 may beperiodic, while in other embodiments the protrusions 4-210 may beaperiodic.

In some implementations, a surface-plasmon structure 4-212 may be spacedfrom a material 2-221 in which the sample well is formed as depicted inFIG. 4-2B. For example, there may be an intervening dielectric layer4-247 between the surface-plasmon structure 4-212 and the material4-230. According to some embodiments, a surface plasmons structure 4-212may be located adjacent a divot 3-216 of a sample well, as depicted inthe drawing. For example, a surface-plasmon structure 4-212 may belocated adjacent sidewalls of a divot 3-216, as depicted in FIG. 4-2B.

FIG. 4-2C illustrates a surface-plasmon structure 4-214 that is formedas a concentric, circular grating. The structure 4-214 may compriseconcentric conducting rings 4-215, according to some embodiments. Therings may be separated by a regular spacing s and have a height h, asdescribed in connection with FIG. 4-2A. According to some embodiments, asample well 4-210 with an optional divot may be located at a center ofthe rings. The circular grating may be patterned adjacent a base of thesample well.

A periodicity of a surface-plasmon structure may be selected to form aresonant structure according to some embodiments. For example a spacings of a surface-plasmon structure may be selected to be approximatelyone-half wavelength of a surface-plasmon wave that is generated in thestructure by the excitation energy. When formed as a resonant structure,a surface-plasmon structure may accumulate and resonate excitationenergy along the direction of the periodic surface-plasmon structure.Such a resonant behavior can intensify electromagnetic energy within asample well, or adjacent a sample well, as depicted in FIG. 4-2D. Whilethe spacing of the surface plasmon structure may be periodic in someembodiments, in other embodiments the spacing may be aperiodic. Usingaperiodic spacing allows the field enhancement to be specificallydesigned for the wavelengths of excitation energy and wavelengths ofemission energy involved. FIG. 4-2D represents numerically simulatedelectromagnetic field results at the base of the sample well and arounda periodic surface-plasmon structure. The surface-plasmon structure4-216 is located adjacent the material 2-221 in which the sample well isformed, and is adjacent a base of a sample well 2-211. Thesurface-plasmon structure may be in the form of a grating or circulargrating that repeats at regular or irregular spacing intervals inregions away from the sample well and outside the simulated region. Forexample, there may be between three and fifty repeated gratingprotrusions of the surface-plasmon structure 4-216. A region of highintensity 4-240 can be seen at the base of the sample well 2-211. Theintensity within this region has been enhanced by more than a factor of2 over the surrounding region just below the surface-plasmon structure.

FIG. 4-2E depicts, in elevation view, an alternative embodiment of aresonant surface-plasmon structure 4-218. According to some embodiments,a surface-plasmon structure may be formed as periodic or aperiodicgrating or grid patterns, and may be patterned in multiple layers 4-247.A sample well 2-211 may be patterned through the multiple layers 4-247and within the resonant surface-plasmon structure 4-218, according tosome embodiments. In some implementations, a resonant surface-plasmonstructure may comprise discrete conductive elements 4-222 is depicted inthe plan view of FIG. 4-2F. In some implementations, a resonantsurface-plasmon structure may comprise a continuous lattice pattern4-250, as depicted in FIG. 4-2G. A dielectric filler 4-252 may belocated in voids of the conductive material 4-250, and a sample well2-211 may be located with a void.

There are a variety of different surface-plasmon structures that may beused to enhance coupling into a sample well or to affect emission from asample within the sample well. FIG.4-2H depicts, in plan view, yet analternative embodiment of the surface-plasmon structure. An elevationview of the structure is depicted in FIG. 4-2I. According to someimplementations, a surface-plasmon structure may comprise an array ofdiscs distributed around a sample well 2-211. In some implementations,instead of using conductive discs 4-260, a surface-plasmon structure maycomprise a conductive layer through which a distributed pattern of holesare formed. Such a structure may be referred to as a “nano-antenna.”

A variety of different processes may be used to pattern surface-plasmonstructures adjacent a sample well. FIG. 4-3A through FIG. 4-5E depictstructures associated with process steps that may be used to formsurface-plasmon structures adjacent to a sample well, according to someembodiments. Referring now to FIG. 4-3A, a process for forming asurface-plasmon structure may comprise forming a resist layer 4-310 onan anti-reflective coating (ARC) 4-320 on a masking layer 4-330. Thelayers may be disposed on a transparent dielectric layer 3-235,according to some implementations. The resist layer 4-310 may comprise aphotoresist or an electron- or ion-beam resist that may belithographically patterned. The masking layer 4-330 may comprise a hardmask formed of an inorganic material (e.g., silicon or silica nitride,or any other suitable material), according to some embodiments.

In some implementations, a photolithographic process may be used topattern the resist 4-310 as depicted in FIG. 4-3B. The selected patternmay comprise a layout of protrusions or holes that will be used to forma desired surface-plasmon structure. After development of the resist4-310, regions of the ARC will be exposed, and the pattern may be etchedinto the ARC layer 4-320 and then into the masking layer 4-330. Theresist and ARC may be stripped from the substrate, and a resultingstructure may appear as shown in FIG. 4-3C. The masking layer 4-330 maythen be used as an etch mask, so that the pattern may be transferredinto the underlying dielectric layer 3-235 via a selective anisotropicetch, as depicted in FIG. 4-3D.

A conductive material 2-221, or a layer of materials comprising aconductor, may then be deposited over the region, as illustrated in FIG.4-3E. Any suitable conductive material may be used for forming a surfaceplasmon structure, whether or not it is deposited as a separate layerfrom the material 2-221. For example, in some cases, a first conductivematerial may be deposited as a base layer of material 2-221 in which asurface-plasmon structure is formed. Examples of materials that may beused for forming a surface-plasmon structure include, but are notlimited to, Au, Al, Ti, TiN, Ag, Cu, and alloys or combination layersthereof.

The material 2-221, or layer of materials, may be deposited by anysuitable deposition process, including but not limited to a physicaldeposition process or a chemical vapor deposition process. In someembodiments, the material 2-221 may have a thickness betweenapproximately 80 nm and approximately 300 nm. In some implementations,the material 2-221 may be planarized (e.g., using a CMP process), thoughplanarization is not necessary. A sample well may be formed in thematerial 2-221 using any suitable process described herein in connectionwith fabricating a sample well.

The inventors have recognized that forming a surface-plasmon structureaccording to the steps shown in FIG. 4-3A through FIG. 4-3E may requireaccurate alignment of the sample well to the surface-plasmon structure.For example, a surface-plasmon structure comprising a concentricgrating, as depicted in FIG. 4-2C, may require accurate alignment of thesample well 2-211 to the center of the surface-plasmon structure 4-214.To avoid fabrication difficulties associated with such accuratealignment, self-alignment processes depicted in FIG. 4-4A through FIG.4-5E may be used.

Referring now to FIG. 4-4A, a process for forming a surface-plasmonstructure and sample well that is self-aligned to the surface-plasmonstructure may comprise forming a masking layer 4-410 on a transparentdielectric layer 2-235. The masking layer may comprise a hard maskformed of an inorganic material, such as silicon or silica nitride,according to some embodiments. A thickness of the masking layer 4-410may be approximately equal to a desired height of a sample well 2-212.For example, the thickness of the masking layer may be betweenapproximately 50 nm and approximately 200 nm, according to someembodiments, though other thicknesses may be used in other embodiments.

The masking layer 4-410 may be patterned to create voids 4-430 havingthe desired pattern of a surface-plasmon structure that will bepatterned in the dielectric layer 2-235. The patterning of the maskinglayer 4-410 may be done with any suitable lithography process (e.g.,photolithography, electron-beam lithography, ion-beam lithography, EUVlithography, x-ray lithography). The resulting structure may appear asshown in FIG. 4-4B. The structure may include a central pillar 4-420,which will be used subsequently to form the self-aligned sample well.

A resist 4-440 (e.g., a photoresist) may then be patterned over thepatterned masking layer 4-410, as depicted in FIG. 4-4C. Alignment forpatterning the resist 4-440 (e.g., mask to substrate alignment) need notbe highly accurate, and only requires the resist 4-440 to cover acentral pillar 4-420 and not cover voids 4-430 that will be used to formthe surface-plasmon structure.

A selective anisotropic etch may then be used to etch the dielectriclayer 2-235 and transfer the pattern of the surface-plasmon structureinto the dielectric, as depicted in FIG. 4-4D according to someembodiments. A selective isotropic etch may then be used to remove theexposed portions of the masking layer 4-410. The isotropic etch may be awet etch, for example, though an isotropic dry etch may be used in someembodiments. Because the resist 4-440 covers the central pillar 4-420,the central pillar will not be etched and remain on the substrate, asdepicted in FIG. 4-4E. The resist 4-440 may then be stripped from thesubstrate exposing the pillar 4-420, as depicted in FIG. 4-4F.

According to some embodiments, a metal conductive material 2-221, or astack of materials including a conductive material, may then bedeposited over the region as illustrated in FIG. 4-4G. The centralpillar 4-420 and a cap of deposited material over the pillar may then beremoved by a selective wet etch of the pillar, lifting off the cap. Theremoval of the central pillar leaves a sample well that is self-alignedto the underlying surface-plasmon structure 4-450.

An alternative process may be used to form a sample well that isself-aligned to a surface-plasmon structure, and is depicted in FIG.4-5A through FIG. 4-5E. According to some embodiments, one or moreconductive layers 4-510, 4-520 may be patterned on a transparentdielectric layer 2-235 using any suitable lithography process, asdepicted in FIG. 4-5A. In some implementations, a first layer 4-510 maycomprise aluminum, and a second layer 4-520 may comprise titaniumnitride, though other material combinations may be used in variousembodiments. A total thickness of the one or more layers may beapproximately equivalent to a desired height of the sample well,according to some embodiments. The patterning may form a sample well2-211, and voids 4-525 adjacent the sample well in the one or more metallayers. The voids may be arranged in the pattern of a desiredsurface-plasmon structure.

In some implementations, the dielectric layer 3-235 may be etched totransfer the pattern of the surface-plasmon structure and sample well2-211 into the dielectric layer, as depicted in FIG. 4-5B. The etchdepth into the dielectric may be between approximately 20 nm andapproximately 150 nm, according to some embodiments. A resist 4-440 maybe patterned to cover the sample well, as depicted in FIG. 4-5C.Alignment for patterning the resist need not be highly accurate, andonly need cover the sample well without covering adjacent etched regionsof the dielectric layer 2-235 that will be used to form thesurface-plasmon structure.

As illustrated in FIG. 4-5D, a conductive material 4-512, or layers ofmaterials including a conductor, may be deposited over the region usingany suitable deposition process. The material 4-512 may fill the etchedregions of the dielectric layer, and may extend above the one or morelayers 4-510, 4-520. The resist 4-440 and the material covering theresist may then be removed according to a lift-off process. Theresulting structure, shown in FIG. 4-5E, leaves a sample well that isself-aligned to the surrounding surface-plasmon structure. The samplewell includes a divot 3-216.

In some embodiments the process depicted in FIG. 4-5A through FIG. 4-5Emay be used to form a sample well that does not have a divot 3-216. Forexample, the resist 4-440 may be patterned over the sample well 2-211before the dielectric layer 2-235 is etched. The dielectric layer 2-235may then be etched, which will transfer the pattern of thesurface-plasmon structure to the dielectric layer but not form a divot.The process may then proceed as illustrated in FIG. 4-5D and FIG. 4-5Eto create a self-aligned sample well having no divot.

Other structures, in addition to or as an alternative to surface-plasmonstructures, may be patterned in the vicinity of the sample well 2-211 toincrease the excitation energy within the sample well. For example somestructures may alter the phase and/or the amplitude of the incidentexcitation field so as to increase the intensity of the excitationenergy within the sample well. FIG. 4-6A depicts a thin lossy film 4-610that may be used to alter the phase and amplitude of incident excitationenergy and increase the intensity of electromagnetic radiation withinthe sample well.

According to some embodiments, a thin lossy film may create constructiveinterference of the excitation energy, resulting in field enhancementwithin an excitation region of the sample well. FIG. 4-6B depicts anumerical simulation of excitation energy incident upon a sample wellwhere a thin lossy film 4-610 has been formed immediately adjacent thesample well. For the simulation, the sample well has a diameter ofapproximately 80 nm and is formed in a metallic layer of goldapproximately 200 nm thick. The sample well comprises an SCN, andsuppresses propagation of excitation energy through the sample well. Thethin lossy film 4-610 is approximately 10 nm thick, is formed fromgermanium, and covers an underlying transparent dielectric comprisingsilicon dioxide. The thin lossy film extends across an entrance apertureof the sample well. The simulation shows that the intensity of theexcitation energy is a highest value at the entrance aperture of thesample well. The intensity of the excitation energy in this brightregion 4-620 is more than twice the value of the intensity to the leftand right of the sample well.

A thin lossy film may be made from any suitable material. For example, athin lossy film may be made from a material where the index ofrefraction n is approximately the same order of magnitude as theextinction coefficient k for the material. In some embodiments, a thinlossy film may be made from a material where the index of refraction nis within about two orders of magnitude difference from the value of theextinction coefficient k of the material. Non-limiting examples of suchmaterials at visible wavelengths are germanium and silicon.

A thin lossy film may be any suitable thickness, which may depend upon acharacteristic wavelength, or wavelengths, associated with theexcitation source, or sources. In some embodiments, a thin lossy filmmay be between approximately 1 nm and approximately 45 nm thick. Inother embodiments, a thin lossy film may be between approximately 15 nmand approximately 45 nm thick. In still other embodiments, a thin lossyfilm may be between approximately 1 nm and approximately 20 nm thick.

Effects of a thin lossy film on reflectance from the material 2-221 inwhich a sample well is formed, excitation energy loss within the thinlossy film, and excitation energy loss within the material 2-221 areshown in the graph of FIG. 4-6C. One curve plotted in the graphrepresents a reflectance curve 4-634, and shows how reflectance from thematerial 2-221 and the thin lossy film 4-610 vary as the thickness ofthe thin lossy film changes from 0 nm to 100 nm. The reflectance reachesa minimum value at about 25 nm, according to the simulated embodiment.The reflectance minimum will occur at different thicknesses depending ona characteristic wavelength of the excitation energy and materials usedfor the thin lossy film and material 2-221. In some implementations athickness of thin lossy film is selected such that the reflectance isapproximately at its minimal value.

In some embodiments, a thin lossy film 4-610 may be spaced from a samplewell 2-211 and material 2-221, as depicted in FIG. 4-6D. For example, athin dielectric layer 4-620 (e.g., a silicon oxide SiOx) may be formedover a thin lossy film, and a sample well 2-211 may be formed adjacentthe dielectric layer 4-620. A thickness of the dielectric layer 4-620may be between approximately 10 nm and approximately 150 nm according tosome embodiments, though other thicknesses may be used in someembodiments.

Although depicted as a single layer, a thin lossy film may comprisemultiple layers of two or more materials. In some implementations, amultilayer stack comprising alternating layers of a thin lossy film4-610 and a dielectric layer 4-620 may be formed adjacent a sample well2-211, as depicted in FIG. 4-6E. A thickness of a thin lossy film 4-610in a stack of layers may be between approximately 5 nm and approximately100 nm, and a thickness of a dielectric layer 4-620 within the stack maybe between approximately 5 nm and approximately 100 nm, according tosome embodiments. In some implementations, the multilayer stack maycomprise a layer of silicon dioxide (4.2 nm thick), a layer of silicon(14.35 nm thick), and a layer of germanium (6.46 nm thick), though otherthicknesses may be used in other embodiments. In some implementations,the multilayer stack may comprise a layer of silicon dioxide(approximately 4.2 nm thick), a layer of silicon (approximately 14.4 nmthick), and a layer of germanium (approximately 6.5 nm thick), thoughother thicknesses may be used in other embodiments.

A thin lossy film may be fabricated from any suitable material thatexhibits at least some loss to the incident radiation. In someembodiments, a thin lossy film may comprise a semiconductor material,for example silicon and germanium, though other materials may be used.In some implementations, a thin lossy film may comprise inorganicmaterial or a metal. In some embodiments, a thin lossy film may comprisean alloy or compound semiconductor. For example, a thin lossy film maycomprise an alloy including Si (57.4% by weight), Ge (25.8% by weight),and SiO2 (16.8% by weight), though other ratios and compositions may beused in other embodiments.

According to some embodiments, a thin lossy film may be formed on thesubstrate using any suitable blanket deposition process, for example, aphysical deposition process, a chemical vapor deposition process, a spinon process, or a combination thereof. In some embodiments, a thin lossyfilm may be treated after deposition, e.g., baked, annealed and/orsubjected to ion implantation.

Other phase/amplitude altering structures may be used additionally oralternatively to enhance excitation energy within the sample well.According to some implementations and as shown in FIG. 4-7A, areflective stack 4-705 may be spaced from a sample well 2-211. In someembodiments, a reflective stack may comprise a dielectric stack ofmaterials having alternating indices of refraction. For example a firstdielectric layer 4-710 may have a first index of refraction, and asecond dielectric layer 4-720 may have a second index of refractiondifferent than the first index of refraction. The reflective stack 4-705may exhibit a high reflectivity for excitation energy in someembodiments, and exhibit a low reflectivity for radiative emission froman emitter within the sample well. For example, a reflective stack 4-705may exhibit a reflectivity greater than approximately 80% for excitationenergy and a reflectivity lower than approximately 40% for emission froma sample, though other reflectivity values may be used in someembodiments. A dielectric layer 4-730 that transmits the excitationenergy may be located between the reflective stack and the sample well.

According to some implementations, a reflective stack 4-705 depicted inFIG. 4-7A may form a resonator with the material 2-221 in which thesample well 2-211 is formed. For example, the reflective stack may bespaced from the material 2-221 by a distance that is approximately equalto one-half the wavelength of the excitation energy within thedielectric material 4-730, or an integral multiple thereof. By forming aresonator, excitation energy may pass through the reflective stack,resonate, and build up in the space between the material 2-221 and thereflective stack 4-705. This can increase excitation intensity withinthe sample well 2-211. For example, the intensity may increase withinthe resonant structure by more than a factor of 2 in some embodiments,and more than a factor of 5 in some embodiments, and yet more than afactor of 10 in some embodiments.

Additional structures may be added in the vicinity of the sample well,as depicted in FIG. 4-7B and FIG. 4-7C. According to some embodiments, adielectric plug 4-740 having a first index of refraction that is higherthan a second index of refraction of the dielectric layer 4-730 may beformed adjacent the sample well 2-211, as depicted in FIG. 4-7B. Theplug may be in the shape of a cylinder having a diameter approximatelyequal to that of the sample well, though other shapes and sizes may beused. Because of its higher refractive index, the dielectric plug 4-740may condense and guide excitation energy toward the sample well.

A dielectric structure, such as the plug 4-740, may be used with orwithout a reflective stack 4-705, according to some embodiments. Such adielectric structure may be referred to as a dielectric resonantantenna. The dielectric resonant antenna may have any suitable shape,for example, cylindrical, rectangular, square, polygon old, trapezoidal,or pyramid.

FIG. 4-7C and FIG. 4-7D depict a photonic bandgap (PBG) structure thatmay be formed in the vicinity of a sample well 2-211, according to someembodiments. A photonic bandgap structure may comprise a regular arrayor lattice of optical contrast structures 4-750. The optical contraststructures may comprise dielectric material having a refractive indexthat is different from a refractive index of the surrounding dielectricmaterial, according to some embodiments. In some implementations, theoptical contrast structures 4-750 may have a loss value that isdifferent from the surrounding medium. In some implementations, a samplewell 2-211 may be located at a defect in the lattice as depicted in FIG.4-7D. According to various embodiments, the defect in the photoniclattice may confine photons within the region of the defect can enhancethe intensity of the excitation energy at the sample well. Theconfinement due to the photonic bandgap structure may be substantiallyin two dimensions transverse to a surface of the substrate. Whencombined with the reflective stack 4-705, confinement may be in threedimensions at the sample well. In some embodiments, a photonic bandgapstructure may be used without a reflective stack.

Various methods have been contemplated for fabricating theexcitation-coupling structures depicted in FIG. 4-6A through FIG. 4-7D.Structures that require thin planar films (e.g., dielectric films ofalternating refractive index) may be formed by planar depositionprocesses, according to some embodiments. Planar deposition processesmay comprise physical deposition (for example, electron beam evaporationor sputtering) or chemical vapor deposition processes. Structures thatrequire discrete embedded dielectrics formed in three-dimensionalshapes, such as a dielectric resonant antenna 4-740 shown in FIG. 4-7Bor the optical contrast structures 4-750 shown in FIG. 4-7C, may beformed using lithographic patterning and etching processes to etch thepattern into the substrate, and using subsequent deposition of adielectric layer, and a planarization of the substrate, for example.Also contemplated are self-alignment processing techniques for formingdielectric resonant antennas as well as photonic bandgap structures inthe vicinity of the sample well 2-211.

FIG. 4-8A through FIG. 4-8G depict structures associated with processsteps for just one self-alignment process that may be used to form aphotonic bandgap structure and a self-aligned sample well as illustratedin FIG. 4-7C. According to some embodiments, a reflective stack 4-705may be first formed on a substrate above a dielectric layer 3-235, asillustrated in FIG. 4-8A.

A second dielectric layer 4-730 may then be deposited over thereflective stack. The thickness of the dielectric layer 4-730 may beapproximately equal to about one-half a wavelength of the excitationenergy in the material, or an integral multiple thereof. Process stepsdescribed in connection with FIG. 4-4A through FIG. 4-4E may then becarried out to form a pillar 4-420 above the dielectric layer 4-730 anda pattern of etched features 4-810 for the photonic bandgap structure.The etched features may extend into the dielectric layer 4-730 andoptionally into the reflective stack 4-705. The resulting structure mayappear as shown in FIG. 4-8A.

A resist 4-440 covering the pillar 4-420 may be stripped from thesubstrate and a conformal deposition performed to fill the etchedfeatures with a filling material 4-820, as depicted in FIG. 4-8B. Thefilling material 4-820 may be the same material that is used to form thepillar 4-420, according to some embodiments. For example the fillingmaterial 4-820 and the pillar 4-420 may be formed of silicon nitride andthe dielectric layer 4-730 may comprise an oxide, e.g., SiO2.

An anisotropic etch may then be carried out to etch back the fillingmaterial 4-820. The filling material may be etched back to expose asurface of the dielectric layer 4-730, according to some embodiments,resulting in a structure as depicted in FIG. 4-8C. The etch may leave apillar 4-830 comprising the original pillar 4-420 and sidewalls 4-822that remain from the filling material 4-820.

A resist 4-440 may then be patterned over the substrate as depicted inFIG. 4-8D. For example, the resist may be coated onto the substrate, ahole patterned in the resist, and the resist developed to open up aregion in the resist around the pillar 4-830. Alignment of the hole tothe pillar need not be highly accurate, and only need expose the pillar4-830 without exposing the underlying photonic bandgap structuresembedded in the dielectric layer 4-730.

After the pillar 4-830 is exposed, and isotropic etch may be used toreduce the transverse dimension of the pillar. According to someembodiments, the resulting pillar shape may appear as depicted in FIG.4-8E. The resist 4-440 may then be stripped from the substrate and amaterial 2-221, or layers of materials, may be deposited over theregion. In some embodiments, the material 2-221 may be etched back usinga CMP process to planarize the region as depicted in FIG. 4-8F.Subsequently, a selective dry or wet etch may be used to remove theremaining pillar structure leaving a sample well 2-211, as illustratedin FIG. 4-8G. As indicated by the drawings, the sample well 2-211 isself-aligned to the photonic bandgap structure patterned in thedielectric layer 4-730.

As an alternative process, the filling material 4-820 may comprise adifferent material than the material used to form the pillar 4-420. Inthis process, the steps associated with FIG. 4-8D and FIG. 4-8E may beomitted. After deposition of material 2-221 and planarization, asdepicted in FIG. 4-8F, a selective etch may be performed to remove thepillar 4-420. This may leave sidewalls of the filling material 4-820lining the sample well 2-211.

D. Non-Radiative Excitation-Coupling Structures

The present disclosure provides structures for non-radiative coupling ofexcitation energy to a sample within the sample well. Just oneembodiment of a non-radiative coupling structure is depicted in FIG.4-9A. According to some embodiments, a non-radiative coupling structuremay comprise a semiconductor layer 4-910 formed immediately adjacent asample well 2-211. The semiconductor layer 4-910 may be an organicsemiconductor in some embodiments, or an inorganic semiconductor in someembodiments. In some implementations, a divot 3-216 may, or may not, beformed in the semiconductor layer. The semiconductor layer 4-910 mayhave a thickness between approximately 5 nm and approximately 100 nmaccording to some embodiments, though other thicknesses may be used insome embodiments. According to some implementations, excitation energyor photons 4-930 from an excitation source may impinge upon thesemiconductor layer 4-910 and produce excitons 4-920. The excitons maydiffuse to a surface of the sample well where they may non-radiativelyrecombine and transfer energy to a sample adjacent the walls of thesample well.

FIG. 4-9B depicts another embodiment in which a semiconductor layer4-912 may be used to non-radiatively transfer energy from excitationenergy to a sample. In some embodiments, a semiconductor layer 4-912 maybe formed at the bottom of a sample well or in a divot of the samplewell 2-211, as depicted in the drawing. The semiconductor layer 4-912may be formed in a sample well by using a directional deposition processas described herein in connection with process steps for depositing anadherent at the base of the sample well, according to some embodiments.The semiconductor layer 4-912 may have a thickness between approximately5 nm and approximately 100 nm according to some embodiments, thoughother thicknesses may be used in other embodiments. Incident radiationmay generate excitons within the semiconductor layer, which may thendiffuse to the a bottom surface of the sample well 2-211. The excitonsmay then non-radiatively transfer energy to a sample within the samplewell.

The present disclosure also provides multiple non-radiative pathways fortransferring excitation energy to a sample. According to someembodiments, and as depicted in FIG. 4-9C, an energy-transfer particle4-940 may be deposited within a sample well. The energy-transferparticle may comprise a quantum dot in some embodiments, or may comprisea molecule in some embodiments. In some implementations, theenergy-transfer particle 4-940 may be functionalized to a surface of thesample well through a linking molecule. A thin semiconductor layer 4-910may be formed adjacent the sample well, or within the sample well, andexcitons may be generated within the semiconductor layer from theexcitation energy incident upon the semiconductor layer, as depicted inthe drawing. The excitons may diffuse to the surface of the sample well,and non-radiatively transfer energy to the energy-transfer particle4-940. The energy-transfer particle 4-940 may then non-radiativelytransfer energy to a sample 3-101 within the sample well.

According to some implementations, there may be more than oneenergy-transfer particle 4-940 within a sample well. For example, alayer of energy-transfer particles 4-942 may be deposited within asample well, such as the sample well depicted in FIG. 4-9C.

In some implementations, energy-transfer particles 4-942, or a singleenergy-transfer particle 4-940, may be deposited at a base of a samplewell, as depicted in FIG. 4-9D. The energy-transfer particle, orparticles, may radiatively or non-radiatively transfer excitation energyto a sample 3-101 within the well. For example, an energy-transferparticle may absorb incident energy to form an excited state of theenergy-transfer particle, and then radiatively or non-radiativelytransfer energy to the sample 3-101.

In some implementations, an energy-transfer particle may absorb incidentexcitation energy, and then re-emit radiative energy at a wavelengththat is different than the wavelength of the absorbed excitation energy.The re-emitted energy may then be used to excite a sample within thesample well. FIG. 4-9E represents spectral graphs associated with adown-converting energy-transfer particle. According to some embodiments,a down-converting energy-transfer particle comprises a quantum dot thatmay absorb short wavelength radiation (higher energy), and emit one ormore longer wavelength radiations (lower energy). An example absorptioncurve 4-952 is depicted in the graph as a dashed line for a quantum dothaving a radius between 6 to 7 nm. The quantum dot may emit a first bandof radiation illustrated by the curve 4-954, a second band of radiationillustrated by the curve 4-956, and a third band of radiationillustrated by the curve 4-958.

In some implementations an energy-transfer particle may up convertenergy from an excitation source. FIG. 4-9F depicts spectra associatedwith up conversion from an energy-transfer particle. According to someembodiments, a quantum dot may be excited with radiation atapproximately 980 nm, and then re-emit into one of three spectral bandsas illustrated in the graph. A first band may be centered atapproximately 483 nm, a second band may be centered at approximately 538nm, and a third band may be centered at approximately 642 nm. There-emitted photons from the quantum dot are more energetic than thephotons of the radiation used to excite the quantum dot. Accordingly,energy from the excitation source is up-converted. One or more of theemitted spectral bands may be used to excite one or more one or moresamples within the sample well.

E. Directing Emission Energy Towards the Sensor

The assay chip 2-110 may include one or more components per pixel toimprove collection of emission energy by the sensors on the instrument.Such components may be designed to spatially direct emission energytowards the sensors and increase the directionality of the emissionenergy from the sample well 2-211. Both surface optics and far-fieldoptics may be used to direct the emission energy towards the sensor.

1. Surface Optics

Components within a pixel of the assay chip 2-110 located near thesample well of the pixel may be configured to couple with the emissionenergy emitted by a sample. Such components may be formed at theinterface between two layers of the assay chip. For example, someemission energy coupling elements may be formed at the interface betweena sample well layer and the layer adjacent to the sample well layeropposite to where the sample wells are formed. In some instances, thelayer underneath the sample well layer is a dielectric layer and theemission energy coupling elements may support surface plasmons. In otherembodiments, the sample well layer may be a conductive material adjacentto an optically-transparent material. Surface-energy coupling elementsmay be surface optical structures that are excited by and interact withradiative emission from the sample well.

A characteristic dimension of a surface optical structure such as agrating period, feature size, or distance from the sample well may beselected to maximally couple a parallel component of an emission energymomentum vector into a surface wave momentum vector for a surfaceplasmon. For example, the parallel component of the emission energymomentum vector may be matched to the surface wave momentum vector for asurface plasmon supported by the structure, according to someembodiments. In some embodiments, a distance d from the sample well toan edge or characteristic feature of a surface optical structure may beselected so as to direct emission energy from the sample well in aselected direction, such as normal to the surface or inclined at anangle θ from normal to the surface. For example, the distance, d, may bean integral number of surface-plasmon wavelengths for directing emissionnormal to the surface. In some embodiments, distance, d, may be selectedto be a fractional surface-plasmon wavelength, or wavelength modulothereof.

According to some embodiments, the surface optical structures may directradiative emission energy from a sample well in a direction normal tothe sample well layer. The coupled energy may be directed in the normaldirection in a narrowed, directional radiation pattern.

An example of a surface optical structure is a concentric grating. Aconcentric grating structure that may be formed in a pixel of the assaychip to direct emission energy towards one or more sensors of the pixel.The concentric grating structure may be formed around a sample well. Anexample of a concentric circular grating surface 5-102 as a surfaceplasmon structure is depicted in FIG. 5-1. The circular grating maycomprise any suitable number of rings and the number of rings (six)shown in FIG. 10-1 is a non-limiting example. The circular grating maycomprise protruding rings from a surface of a conductive layer. Forexample, the circular grating may be formed at the interface of thesample well layer and a dielectric layer formed underneath the samplewell layer. The sample well layer may be a conductive material and theconcentric grating may be formed by patterning the grating structure atthe interface between the conductive material and the dielectric. Therings of the circular grating may be on a regular periodic spacing, ormay have irregular or aperiodic spacings between the rings. The samplewell may be located at or near the center of the circular grating. Insome embodiments, the sample well may be located off-center to thecircular grating and may be positioned a certain distance from thecenter of the grating. In some embodiments, a grating-type surfaceenergy-coupling component may comprise a spiral grating. An example of aspiral grating 5-202 is depicted in FIG. 5-2. The spiral grating 5-202may comprise a spiral aperture in a conductive film. Any suitabledimensions of the spiral grating may be used to form the spiral grating.

FIG. 5-3 illustrates a radiation pattern 5-302 for emission energy fromthe sample well 2-211. The concentric grating structure 2-223 causes theemission energy to have greater directionality compared to the radiationpattern formed in the absence of the grating structure 2-223. In someembodiments, the emission energy is directed downward, normal to themetal layer 2-221.

Another example of a surface optic or surface plasmon structure is anano-antenna structure. A nano-antenna structure may be designed tospatially direct emission energy from the sample well. In someembodiments, the location of the sample well with respect to thenano-antenna structure is selected so as to direct the emission energyfrom the sample well in a particular direction towards one or moresensors. Nano-antennas may comprise nano-scale dipole antenna structuresthat are designed to produce a directional radiation pattern whenexcited by emission energy. The nano-antennas may be distributed arounda sample well. The directional radiation pattern may result from asummation of the antennas' electromagnetic fields. In some embodiments,the directional radiation pattern may result from a summation of theantennas' electromagnetic fields with the field emitted directly fromthe sample. In some implementations, the field emitted directly from thesample may be mediated by a surface plasmon between the sample well andnano-antenna structure.

The dimensions of the individual nano-antennas that form thenano-antenna structure may be selected for the combined ability of theoverall nano-antenna structure to produce specific distributionpatterns. For example, the diameters of the individual nano-antennas mayvary within a nano-antenna structure. However, in some instances, thediameters may be the same within a set of nano-antennas. In otherimplementations, a few selected diameters may be used throughout theoverall nano-antenna structure. Some nano-antennas may be distributed ona circle of radius R and some may be shifted in a radial direction fromthe circle. Some nano-antennas may be equally spaced around a circle ofradius R (e.g., centered on equivalent polar-angle increments), and somemay be shifted from equal spacing around the circle. In someembodiments, the nano-antennas may be arranged in a spiral configurationaround a sample well. Additionally or alternatively, otherconfigurations of nano-antennas are possible, such as a matrix arrayaround the sample well, a cross distribution, and star distributions.Individual nano-antennas may be shapes other than a circle, such assquare, rectangular, cross, triangle, bow-tie, annular ring, pentagon,hexagon, polygons, etc. In some embodiments, the circumference of anaperture or disc may be approximately an integer multiple of afractional wavelength, e.g., (N/2)λ.

A nano-antenna array may direct emission energy from a sample intoconcentrated radiation lobes. When a sample emits energy, it may excitesurface plasmons that propagate from the sample well to thenano-antennas distributed around the sample well. The surface plasmonsmay then excite radiation modes or dipole emitters at the nano-antennasthat emit radiation perpendicular to the surface of the sample welllayer. The phase of an excited mode or dipole at a nano-antenna willdepend upon the distance of the nano-antenna from the sample well.Selecting the distance between the sample well and an individualnano-antenna controls the phase of radiation emitted from thenano-antenna. The spatial radiation mode excited at a nano-antenna willdepend upon the geometry and/or size of the nano-antenna. Selecting thesize and/or geometry of an individual nano-antenna controls the spatialradiation mode emitted from the nano-antenna. Contributions from allnano-antennas in the array and, in some instances the sample well, maydetermine an overall radiation lobe or lobes that form the radiationpattern. As may be appreciated, phase and spatial radiation mode emittedfrom an individual nano-antenna may depend upon wavelength, so that theoverall radiation lobe or lobes that form the radiation pattern willalso be dependent upon wavelength. Numerical simulations of theelectromagnetic fields may be employed to determine overall radiationlobe patterns for emission energies of different characteristicwavelengths.

The nano-antenna may comprise an array of holes or apertures in aconductive film. For example, the nano-antenna structure may be formedat the interface between a conductive sample well layer and anunderlying dielectric layer. The holes may comprise sets of holesdistributed in concentric circles surrounding a central point. In someembodiments, a sample well is located at the central point of the array,while in other embodiments the sample well may be off-center. Eachcircularly-distributed set of holes may comprise a collection ofdifferent diameters arranged smallest to largest around the circulardistribution. The hole diameters may be different between the sets(e.g., a smallest hole in one set may be larger than a smallest hole inanother set), and the location of the smallest hole may be oriented at adifferent polar angle for each set of circles. In some embodiments,there may be one to seven sets of the circularly-distributed holes in anano-antenna. In other embodiments, there may be more than seven sets.In some embodiments, the holes may not be circular, but may be anysuitable shape. For example, the holes may be ellipses, triangles,rectangles, etc. In other embodiments, the distribution of holes may notbe circular, but may create a spiral shape.

FIGS. 5-4A and 5-4B illustrate an exemplary nano-antenna structurecomprised of holes or apertures in a conductive layer. FIG. 5-4A shows atop planar view of the surface of an assay chip with a sample well 5-108surrounded by holes 5-122. The nano-antenna holes are distributed withtheir centers approximately around a circle of radius R. In thisnon-limiting example, the hole diameters vary by incrementallyincreasing around the circumference of the circle of holes. FIG. 5-4Bshows a schematic of a cross-sectional view of the assay chip shown inFIG. 5-4A along line B-B′. The sample well layer 5-116 that includessample well 5-108 and apertures 5-122 that are part of the nano-antennastructure. Layer 5-118 of the assay chip lies underneath sample welllayer 5-116. Layer 5-118 may be a dielectric material and/or anoptically transparent material.

In some embodiments, the nano-antenna structure may comprise a pluralityof disks. The disks of the nano-antenna structure may be formed asconductive disks protruding from a surface of a conductive material. Theconductive material may be adjacent an optically-transparent material.In some embodiments, the nano-antennas may be distributed around asample well. In some instances, the nano-antennas may be distributedapproximately around a sample well at a circle of radius R. Anano-antenna array may comprise multiple sets of nano-antennasdistributed approximately on additional circles of different radiiaround a sample well.

FIGS. 5-5A and 5-5B illustrate an exemplary embodiment of a nano-antennastructure comprising disks protruding from a conductive layer. FIG. 5-5Ashows a top planar view schematic of the surface of an assay chip with asample well 5-208 surrounded by disks 5-224. The nano-antenna disks aredistributed approximately around a circle of radius R. In thisnon-limiting example, two diameters are used for the disks and the disksalternate between these two diameters around the circumference of thecircle of nano-antenna. FIG. 5-5B shows a schematic of a cross-sectionalview of the assay chip shown in FIG. 5-5A along line C-C′. The samplewell layer 5-216 that includes sample well 5-208 and disks 5-224 thatare part of the nano-antenna structure. The disks 5-224 protrude fromthe sample well layer 5-216 by a certain distance. In some embodiments,the distance the disks extend from the sample well layer may vary withina nano-antenna structure. Layer 5-218 of the assay chip lies underneathsample well layer 5-216. Layer 5-18 may be a dielectric material and/oran optically transparent material. The sample well layer 5-216 and theprotruding disks may be a conductive material.

2. Far Field Optics

In some embodiments, the layer directly under the surface optics may bea spacer layer 2-225 of any suitable thickness and be made of anysuitable dielectric material. The spacer layer may be, for example, 10μm in thickness and may be made of silicon dioxide. Alternatively, thisspacer layer may be 48 μm or 50 μm. The under the spacer layer may beone or more lens layers with additional spacer layers. For example, FIG.5-6A illustrates an upper lens layer 5-601 which may include at leastone refractive lens. In some embodiments, the upper lens layer may belocated 5μm below the sample well layer 2-221. There may be one or morelenses associated with each sample well. In some embodiments, a lensarray may be used. In some embodiments, each lens of the upper lenslayer 5-601 is centered below sample well 2-211 and may have a radius,for example, smaller than 10.5 μm. The upper lens layer may be made ofany suitable dielectric material such as, by way of example and notlimitation, silicon nitride.

The layer directly under the upper lens layer may be a structural and/oroptical layer 5-605 made of any suitable dielectric. This structuraland/or optical layer 5-605 may be made of silicon dioxide in the form offused silica. The layer directly under the structural layer may be alower lens layer 5-603 which may include at least one additional lens.In some embodiments, each lens in the lower lens layer 5-603 may also becentered below the sample well. The lower lens layer 5-603 may be madeof any suitable dielectric material such as, by way of example and notlimitation, silicon nitride. The distance from the top of the upper lenslayer to the bottom of the lower lens layer may be 100-500 μm. The layerdirectly under the lower lens layer may include an anti-reflection layerthat passes both excitation energy and the emission energy and reducesthe amount of light reflected. The layer directly under theanti-reflection layer may include structural components to allow thechip to align with and mount onto the instrument. The layer directlyunder the chip-mounting layer may include a protective cover to protectthe system from damage and contamination, including dust.

While FIG. 5-6A illustrates two lens layers using refractive lenses, anysuitable lens may be used. For example, Fresnel lenses, microlenses,refractive lens pairs and/or flat lenses may be used. FIG. 5-6Billustrates an embodiment using Fresnel lenses in both an upper lenslayer 5-611 and a lower lens layer 5-613, separated by a structuraland/or optical layer 5-605.

In some embodiments, any of the interfaces between the layers describedabove in the chip may include an anti-reflection coating oranti-reflection layer. Both the upper lens layer and the second lenslayer may be arranged below the sample well to focus the luminescenceemitted from the array of sample wells into a relay lens of theinstrument.

IV. Instrument Components

I. Microscopy Layer of the Instrument

In some embodiments, the instrument may include a microscopy layer whichmay include sub-layers as illustrated in FIG. 6-1. In particular, themicroscopy layer may include a sub-layer that includes a polychroicmirror 2-230 tilted at an angle θ to direct the excitation energy towardthe assay chip. This polychroic mirror may be substantially dielectric,and reflects the excitation energy while substantially transmitting theemission energy from the sample in one or more of the sample wells onthe assay chip. Optionally, an astigmatism compensation element 6-101that includes an additional dielectric layer may be provided underneaththe polychroic mirror and tilted at the same angle θ, but about an axisthat is orthogonal to that of the polychroic mirror's tilt, to providecompensation for astigmatism introduced by the polychroic mirror. InFIG. 6-1, the astigmatism compensation element 6-101 is illustrated astilted in the same plane as the top filter, but it should be appreciatedthat the illustration represents a tilting with respect to the topfilter and it is not meant to limit the orientation of the astigmatismcompensation element 6-101 in any way. This astigmatism compensationelement 6-101 may also provide additional filtering. For example, theastigmatism compensation element 6-101 may be another polychroic mirrorthat further filters the excitation energy while transmitting theemission energy. A lens 6-103 may be provided underneath the astigmatismcompensation element 6-101 to further help process the emission energyfrom the sample wells. The lens 6-103 may be, for example, 25.4 μm indiameter, but any suitable diameter may be used. In some embodiments,the lens is a relay lens comprising a plurality of lens elements. Forexample, the relay lens may include six separate lens elements. In someembodiments, the relay lens may be, approximately 17.5 mm in length.Additional filtering elements may be used before or after lens 6-103 tofurther reject the excitation energy to prevent it from reaching thesensors.

A. Sensor chip

Emission energy emitted from a sample in the sample well may betransmitted to the sensor of a pixel in a variety of ways, some examplesof which are described in detail below. Some embodiments may use opticaland/or plasmonic components to increase the likelihood that light of aparticular wavelength is directed to an area or portion of the sensorthat is dedicated to detecting light of that particular wavelength. Thesensor may include multiple sub-sensors for simultaneously detectingemission energy of different wavelengths.

FIG. 6-2A is a schematic diagram of a single pixel of the sensor shipaccording to some embodiments where at least one sorting element 6-127is used to direct emission energy of a particular wavelength to arespective sub-sensor 6-111 through 6-114. The emission energy 2-253travels from a sample well through the assay chip and the optical systemof the instrument until it reaches a sorting element 6-127 of the sensorchip. The sorting element 6-127 couples the wavelength of the emissionenergy 2-253 to a spatial degree of freedom, thereby separating theemission energy into its constituent wavelength components, referred toas sorted emission energy. FIG. 6-2A illustrates schematically theemission energy 2-253 being split into four sorted emission energy pathsthrough a dielectric material 6-129, each of the four paths associatedwith a sub-sensor 6-111 through 6-114 of the pixel. In this way, eachsub-sensor is associated with a different portion of the spectrum,forming a spectrometer for each pixel of the sensor chip.

Any suitable sorting element 6-127 may be used to separate the differentwavelengths of the emission energy. Embodiments may use optical orplasmonic elements. Examples of optical sorting elements include, butare not limited to, holographic gratings, phase mask gratings, amplitudemask gratings, and offset Fresnel lenses. Examples of plasmonic sortingelements include, but are not limited to phased nano-antenna arrays, andplasmonic quasi-crystals.

FIG. 6-2B is a schematic diagram of a single pixel of the sensor chipaccording to some embodiments where filtering elements 6-121 through6-124 are used to direct emission energy of a particular wavelength to arespective sub-sensor and prevent emission energy of other wavelengthsfrom reaching the other sub-sensors. The emission energy 2-253 travelsfrom a sample well through the assay chip and the optical system of theinstrument until it reaches one of the filtering elements 6-121 through6-124. The filtering elements 6-121 through 6-124, each associated witha particular sub-sensor 6-11 through 6-114, are each configured totransmit emission energy of a respective wavelength and reject emissionenergy of other wavelengths by absorbing the emission energy (notillustrated in FIG. 6-1B) and/or reflecting the emission energy. Afterpassing through a respective filtering element, the filtered emissionenergy travels through a dielectric material 6-129 and impinges on acorresponding sub-sensor 6-111 through 6-114 of the pixel. In this way,each sub-sensor is associated with a different portion of the spectrum,forming a spectrometer for each pixel of the sensor chip.

Any suitable filtering elements may be used to separate the differentwavelengths of the emission energy. Embodiments may use optical orplasmonic filtering elements. Examples of optical sorting elementsinclude, but are not limited to, reflective multilayer dielectricfilters or absorptive filters. Examples of plasmonic sorting elementsinclude, but are not limited to frequency selective surfaces designed totransmit energy at a particular wavelength and photonic band-gapcrystals.

Alternatively, or in addition to the above mentioned sorting elementsand filtering elements, additional filtering elements may be placeadjacent to each sub-sensor 6-11 through 6-114. The additional filteringelements may include a thin lossy film configured to create constructiveinterference for emission energy of a particular wavelength. The thinlossy film may be a single or multi-layer film. The thin lossy film maybe made from any suitable material. For example, the thin lossy film maybe made from a material where the index of refraction n is approximatelythe same order of magnitude as the extinction coefficient k. In otherembodiments, the thin lossy film may be made from a material where theindex of refraction n is within about two orders of magnitude differencefrom the value of the extinction coefficient k of the material.Non-limiting examples of such materials at visible wavelengths aregermanium and silicon.

The thin lossy film may be any suitable thickness. In some embodiments,the thin lossy film may be 1-45 nm thick. In other embodiments, the thinlossy film may be 15-45 nm thick. In still other embodiments, the thinlossy film may be 1-20 nm thick. FIG. 6-3A illustrates an embodimentwhere the thin lossy films 6-211 through 6-214 each have a differentthickness determined at least in part by the wavelength that isassociated with each sub-sensor 6-11 through 6-114. The thickness of thefilm determines, at least in part, a distinct wavelength that willselectively pass through the thin lossy film to the sub-sensor. Asillustrated in FIG. 6-211, thin lossy film 6-211 has a thickness d1,thin lossy film 6-212 has a thickness d2, thin lossy film 6-213 has athickness d3, and thin lossy film 6-214 has a thickness d4. Thethickness of each subsequent thin lossy film is less than the previousthin lossy film such that d1>d2>d3>d4.

Additionally, or alternatively, the thin lossy films may be formed of adifferent material with a different properties such that emission energyof different wavelengths constructively interfere at each respectivesub-sensor. For example, the index of refraction n and/or the extinctioncoefficient k may be selected to optimize transmission of emissionenergy of a particular wavelength. FIG. 6-3B illustrates thin lossyfilms 6-221 through 6-224 with the same thickness but each thin lossyfilm is formed from a different material. In some embodiments, both thematerial of the thin lossy films and the thickness of the thin lossyfilms may be selected such that emission energy of a desired wavelengthconstructively interferes and is transmitted through the film.

FIG. 6-1 illustrates an embodiment where a combination of diffractiveelements and lenses are used to sort the emission energy by wavelength.A first layer 6-105 of the sensor chip may include a blazed phasegrating. The blazed grating may be blazed, for example, at an angle ϕsubstantially equal to 40 degrees and the line spacing of the blazedgrating (Λ) may be substantially equal to 1.25 μm. One of skill in theart would appreciate that different blaze angles and periodicities maybe used to achieve separation of light of different wavelengths ofemission energy. Moreover, any suitable diffractive optical element maybe used to separate the different wavelengths of the emission energy.For example, a phase mask, an amplitude mask, a blazed grating or anoffset Fresnel lens may be used.

A second layer 6-106 of the sensor chip 2-260 may include one or moreFresnel lenses disposed beneath the first layer 6-105 to further sortand direct the emission energy to the sensors 6-107. Moreover, anysuitable lens element may be used to further separate the differentwavelengths of the emission energy. For example, a refractive lens maybe used instead of a Fresnel lens.

The various components of FIG. 6-1 may be spaced apart at any suitabledistances. For example, the surface of the sensors may be located at adistance of 5 μm beneath the Fresnel lens layer 6-106; the distance fromthe center of the lens 6-103 of the microscopy layer to the Fresnel lenslayer 6-106 may be 50.6 mm; the blazed phase grating 6-105 may belocated at a distance of approximately 100 μm above the surface of thesensors. Alternatively, the distance from the bottom of the assay chipto the top of the grating 6-105 may be approximately 53 mm. The width ofthe sensor layer may be approximately 10 mm.

The various layers of the assay chip and instrument need not be in theorder described above. In some embodiments, the focusing and/or sortingelements and the imaging optics of the instrument may be in reverseorder. For example, the blazed phase grating 6-105 may be placed afterthe Fresnel lens layer 6-106. Alternatively, the focusing and/or sortingelements and the imaging optics may be incorporated into a singlediffractive optical element (DOE). In addition, various components ofthe assay chip and instrument may be intermingled such that, forexample, imaging optics may occur both above and below the focusingand/or sorting elements.

Any of the interfaces between the layers, including the interfacebetween air and a layer of the system, described above in the system mayinclude an anti-reflection coating.

B. Embodiment of the Optical Block of the Instrument

In some embodiments, the optical block of the instrument 1-120 mayinclude some or all of the optical components described above. Theoptical block may provide the optical components as arranged in FIG.6-4. In addition to the components described above, the optical blockmay include a first fiber connector 6-401 where a first optical fibercarrying a first wavelength of excitation energy may connect and asecond fiber connector 6-402 where a second optical fiber carrying asecond wavelength of excitation energy may connect. By way of exampleand not limitation, the first excitation wavelength of the excitationenergy may be 630-640 nm. The optical fiber connectors may be anysuitable conventional connector, such as an FC or an LC connector. Iftwo different wavelengths are input, the wavelengths may be combinedwith a wavelength combiner 6-403, such as a dichroic or polychroicmirror. The second excitation wavelength may be 515-535 nm. The inputexcitation energy may be any suitable polarization, such as linearpolarization. In some embodiments, the fiber carrying the excitationenergy may be a polarization-maintaining fiber. Optionally, excitationfilters and polarizers, such as optical fiber-to-free-space couplers,may be used after the optical fiber input to further filter or modifycharacteristics of the excitation energy.

The optical block may include one or more metal housings to hold lensesand other optical components for optical processing such as beamshaping. FIG. 6-4 illustrates four metal housings 6-405 through 6-408,each holding a lens and/or other optical components. There may be anynumber of lenses used to collimate and focus the excitation energy. Oneor more mirrors 6-411 and 6-412 are situated between some of the metalhousings for guiding the excitation energy towards the assay chip 2-110.In FIG. 6-4, the first mirror 6-411directs the excitation energy fromthe second housing 6-406 to the third housing 6-407 and the secondmirror 6-412 reflects the excitation energy from the fourth housing6-408 to a polychroic dielectric mirror 2-230. The polychroic dielectricmirror 2-230 directs the excitation energy towards an astigmatismcompensation filter 6-601.

In some embodiments, circularly polarized light may be directed into thesample well to cause the luminescent markers to emit luminescence withsimilar strength. A quarter-wave plate may be used to transfer thelinearly polarized light to circularly polarized light before it reachesthe assay chip. The polychroic dielectric mirror 2-230 directs theexcitation energy to the quarter wave plate 6-415. As illustrated inFIG. 6-4, the quarter-wave plate 6-415 may be disposed between theastigmatism compensation filter 6-101 and the assay chip 2-110. Thecircularly polarized excitation energy is then directed towards theplurality of pixels on the assay chip. Excitation energy that is notdirected towards the pixels may be absorbed by a beam dump component6-417. Excitation energy that reaches the sample inside one or moresample wells will cause the sample to emit emission energy, which isdirected toward the sensor 2-260. The emission energy may pass throughoptical components such as polarization optics, the astigmatismcompensating element 6-101, the polychroic mirror 2-230 and a relay lens6-103. The polychroic mirror acts as a filer, which may be, by way ofexample, a notch filter, a spike filter or a cut-off filter. The relaylens 6-103 may image the emission energy toward the sensor. A portion ofthe emission energy may then pass through one or more emission filters6-421 and 6-422, situated above the sensor 2-260, which may furtherfilter the emission energy. In some embodiments, the emission filtersmay be tilted at an angle relative to the incident emission energypropagation direction in order to tune the transmission characteristicsof the filters and/or reduce interference caused by back reflections. Ifthe top filter 6-421 is tilted at an angle θ, the bottom filter 6-422may be tilted at the same angle θ, but about an axis that is orthogonalto that of the top filter's tilt, to ensure no astigmatism is introducedinto the emission radiation beam path.

C. Sensors

The present disclosure provides various embodiments of sensors, sensoroperation, and signal processing methods. According to some embodiments,a sensor 2-122 at a pixel of the sensor chip 2-260 may comprise anysuitable sensor capable of receiving emission energy from one or moretags in the sample well, and producing one or more electrical signalsrepresentative of the received emission energy. In some embodiments, asensor may comprise at least one a photodetector (e.g., a p-n junctionformed in a semiconductor substrate). FIG. 7-1A and FIG. 7-1B depictsone embodiment of a sensor that may be fabricated within a pixel 2-100of a sensor chip.

According to some embodiments, a sensor 2-122 may be formed at eachpixel 2-100 of a sensor chip. The sensor may be associated with a samplewell 2-211 of the assay chip. There may be one or more transparentlayers 7-110 above the sensor, so that emission from the sample well maytravel to the sensor without significant attenuation. The sensor 2-122may be formed in a semiconductor substrate 7-120 at a base of the pixel,according to some embodiments, and be located on a same side of thesample well as the assay chip (not shown).

The sensor may comprise one or more semiconductor junction photodetectorsegments. Each semiconductor junction may comprise a well of a firstconductivity type. For example, each semiconductor junction may comprisean n-type well formed in a p-type substrate, as depicted in the drawing.According to some embodiments, a sensor 2-122 may be arranged as abulls-eye detector 7-162, as depicted in the plan view of FIG. 7-1B. Afirst photodetector 7-124 may be located at a center of the sensor, anda second annular photodetector 7-122 may surround the centerphotodetector. Electrical contacts to the wells may be made throughconductive traces 7-134 formed at a first or subsequent metallizationlevel and through conductive vias 7-132. There may be a region of highlydoped semiconductor material 7-126 at contact regions of the vias. Insome embodiments, a field oxide 7-115 may be formed at surfaces betweenthe photodetectors and may cover a portion of each photodetector. Insome implementations, there may be additional semiconductor devices7-125 (e.g., transistors, amplifiers, etc.) formed within the pixeladjacent to the sensor 2-122. There may be additional metallizationlevels 7-138, 7-136 within the pixel.

In some implementations, a metallization levels 7-136 may extend acrossa majority of the pixel and have an opening centered above thephotodetector 7-124, so that emission from the sample well can reach thesensor. In some cases, a metallization level 7-136 may serve as areference potential or a ground plane, and additionally serve as anoptical block to prevent at least some background radiation (e.g.,radiation from an excitation source or from the ambient environment)from reaching the sensor 2-260.

As depicted in FIG. 7-1A and FIG. 7-1B, a sensor 2-122 may be subdividedinto a plurality of photodetector segments 7-122, 7-124 that arespatially and electrically separated from each other. In someembodiments, segments of a sensor 2-122 may comprise regions ofoppositely-doped semiconductor material. For example, a first chargeaccumulation well 7-124 for a first sensor segment may be formed bydoping a first region of a substrate to have a first conductivity type(e.g., n-type) within the first well. The substrate may be p-type. Asecond charge accumulation well 7-122 for a second sensor segment may beformed by doping a second region of the substrate to have the firstconductivity type within the second well. The first and second wells maybe separated by a p-type region of the substrate.

The plurality of segments of the sensor 2-122 may be arranged in anysuitable way other than a bulls-eye layout, and there may be more thantwo segments in a sensor. For example, in some embodiments, a pluralityof photodetector segments 7-142 may be laterally separated from oneanother to form a stripe sensor 7-164, as depicted in FIG. 7-1C. In someembodiments, a quad (or quadrant) sensor 7-166 may be formed byarranging the segments 7-144 in a quad pattern, as depicted in FIG.7-1D. In some implementations, arc segments 7-146 may be formed incombination with a bulls-eye pattern, as depicted in FIG. 7-1E, to forman arc-segmented sensor 7-168. Another sensor configuration may comprisepie-piece sections, which may include individual sensors arranged inseparate section of a circle. In some cases, sensor segments may bearranged symmetrically around a sample well 2-211 or asymmetricallyaround a sample well. The arrangement of sensor segments is not limitedto only the foregoing arrangements, and any suitable distribution ofsensor segments may be used.

The inventors have found that a quadrant sensor 7-166, pie-sectorsensor, or similar sector sensor can scale to smaller pixel sizes morefavorably than other sensor configurations. Quadrant and sectordetectors may consume less pixel area for a number of wavelengthsdetected and active sensor area.

Sensors may be arranged in various geometric configurations. In someexamples, sensors are arranged in a square configurations or hexagonalconfiguration.

Sensors of the present disclosure may be independently (or individually)addressable. An individually addressable is capable of detecting asignal and providing an output independent of other sensors. Anindividually addressable sensor may be individually readable.

In some embodiments, a stacked sensor 7-169 may be formed by fabricatinga plurality of separated sensor segments 7-148 in a vertical stack, asdepicted in FIG. 7-1F. For example, the segments may be located oneabove the other, and there may, or may not, be insulating layers betweenthe stacked segments. Each vertical layer may be configured to absorbemission energy of a particular energy, and pass emission at differentenergies. For example, a first detector may absorb and detectshorter-wavelength radiation (e.g., blue-wavelength radiation belowabout 500 nm from a sample). The first detector may pass green- andred-wavelength emissions from a sample. A second detector may absorb anddetect green-wavelength radiation (e.g., between about 500 nm and about600 nm) and pass red emissions. A third detector may absorb and detectthe red emissions. Reflective films 7-149 may be incorporated in thestack, in some embodiments, to reflect light of a selected wavelengthband back through a segment. For example, a film may reflectgreen-wavelength radiation that has not been absorbed by the secondsegment back through the second segment to increase its detectionefficiency.

In some embodiments with vertically-stacked sensor segments,emission-coupling components may not be included at the sample well toproduce distinct spatial distribution patterns of sample emission thatare dependent on emission wavelength. Discernment of spectrallydifferent emissions may be achieved with a vertically-stacked sensor7-169 by analyzing the ratio of signals from its stacked segment,according to some embodiments.

In some embodiments, segments of a sensor 2-122 are formed from silicon,though any suitable semiconductor (e.g., Ge, GaAs, SiGe, InP, etc.) maybe used. In some embodiments, a sensor segment may comprise an organicphotoconductive film. In other embodiments, quantum dot photodetectorsmay be used for sensor segments. Quantum dot photodetectors may respondto different emission energies based on the size of the quantum dot. Insome embodiments, a plurality of quantum dots of varying sizes may beused to discriminate between different emission energies or wavelengthsreceived from the sample well. For example, a first segment may beformed from quantum dots having a first size, and a second segment maybe formed from quantum dots having a second size. In variousembodiments, sensors 2-122 may be formed using conventional CMOSprocesses.

As described above, emission-coupling components may be fabricatedadjacent the sample well in some embodiments. The sorting elements 2-243can alter emission from a sample within the sample well 2-211 to producedistinct spatial distribution patterns of sample emission that aredependent on emission wavelength. FIG. 7-2A depicts an example of afirst spatial distribution pattern 7-250 that may be produced from afirst sample at a first wavelength. The first spatial distributionpattern 7-250 may have a prominent central lobe directed toward acentral segment of a bulls-eye sensor 7-162, for example, as shown inFIG. 7-2B. Such a pattern 7-250 may be produced by any suitablediffractive element when the sample emits at a wavelength of about 663nm. A projected pattern 7-252 incident on the sensor may appear asillustrated in FIG. 7-2B.

FIG. 7-2C depicts a spatial distribution pattern 7-260 that may beproduced from a second sample emitting at a second wavelength from thesame sample well, according to some embodiments. The second spatialdistribution pattern 7-260 may comprise two lobes of radiation anddiffer from the first spatial distribution pattern 7-250. A projectedpattern 7-262 of the second spatial distribution pattern 7-260 mayappear as depicted in FIG. 7-2D, according to some embodiments. Thesecond spatial distribution pattern 7-260 may be produced by anysuitable diffractive element when the sample emits at a wavelength ofabout 687 nm.

The segments of a sensor 2-122 may be arranged to detect particularemission energies, according to some embodiments. For example,emission-coupling structures adjacent the sample well and segments of asensor may be designed in combination to increase signal differentiationbetween particular emission energies. The emission energies maycorrespond to selected tags that will be used with the sensor chip. Asan example, a bulls-eye sensor 7-162 could have its segments sizedand/or located to better match the projected patterns 7-260, 7-262 froma sample, so that regions of higher intensity fall more centrally withinactive segments of the sensor. Alternatively or additionally,diffractive elements may be designed to alter the projected patterns7-260, 7-262 so that intense regions fall more centrally within segmentsof the sensor.

Although a sensor 2-122 may comprise two segments, it is possible insome embodiments to discern more than two spectrally-distinct emissionbands from a sample. For example, each emission band may produce adistinct projected pattern on the sensor segments and yield a distinctcombination of signals from the sensor segments. The combination ofsignals may be analyzed to discern and identify the emission band. FIG.7-2E through FIG. 7-2H represent results from numerical simulations ofsignals from a two-segment sensor 2-122 exposed to four distinctemission patterns. As can be seen, each combination of signals from thetwo sensor segments is distinct, and can be used to discriminate betweenemitters at the four wavelengths. For the simulation, because the outerdetector segment of the bulls-eye sensor 7-162 had a larger area, moresignal was integrated for that detector. Additionally, light thatimpinged on an area between the detectors generated carriers that maydrift towards either detector segment and contribute to signals fromboth segments.

In some embodiments, there may be N photodetector segments per pixel,where N may be any integer value. In some embodiments, N may be greaterthan or equal to 1 and less than or equal to 10. In other embodiments, Nmay be greater than or equal to 2 and less than or equal to 5. Thenumber M of discernible sample emissions (e.g., distinct emissionwavelengths from different luminescent tags) that may be detected by theN detectors may be equal to or greater than N. The discernment of Msample emissions may be achieved by evaluating the ratio of signals fromeach sensor segment, according to some embodiments. In someimplementations, the ratio, sum and/or amplitudes of the receivedsignals may be measured and analyzed to determine a characteristicwavelength of emission from the sample well.

In some embodiments, more than one emitter may emit at differentcharacteristic wavelengths in a given time window within a sample well2-211. A sensor 2-122 may simultaneously detect signals from multipleemissions at different wavelengths and provide the summed signal fordata processing. In some implementations, multi-wavelength emission maybe distinguishable as another set of signal values from the sensorsegments (e.g., signal values different from those shown in FIG. 7-2Ethrough FIG. 7-2H). The signal values may be analyzed to discern thatmulti-wavelength emission has occurred and to identify a particularcombination of emitters associated with the emissions.

The inventors have also contemplated and analyzed a bulls-eye sensorhaving four concentric segments. Signals from the segments are plottedin FIG. 7-2I and FIG. 7-2J for the same emission conditions associatedwith FIG. 7-2G and FIG. 7-2H, respectively. The four-segment bulls-eyesensor also shows discernable signals that may be analyzed to identify aparticular emitter within the sample well.

When wavelength filtering is used at each sensor segment, or thespectral separation is high, each segment of a sensor may detectsubstantially only a selected emission band. For example, a firstwavelength may be detected by a first segment, a second wavelength maybe detected by a second segment, and a third wavelength may be detectedby a third segment.

Referring again to FIG. 7-1A, there may be additional electroniccircuitry 7-125 within a pixel 2-100 that may be used to collect andreadout signals from each segment of a sensor 2-122. FIG. 7-3A and FIG.7-3D depict circuitry that may be used in combination with amulti-segment sensor, according to some embodiments. As an example,signal collection circuitry 7-310 may comprise three transistors foreach sensor segment. An arrangement of the three transistors is depictedin FIG. 7-3B, according to some implementations. A signal level at acharge accumulation node 7-311 associated with each segment may be resetby a reset transistor RST, and a signal level for the segment(determined by the amount of charge at the charge accumulation node) maybe read out with a read transistor RD.

The pixel circuitry may further include amplification and correlateddouble-sampling circuitry 7-320, according to some embodiments. Theamplification and double-sampling circuitry may comprise transistorsconfigured to amplify signals from the sensor segments as well astransistors configured to reset the voltage level at thecharge-accumulation node and to read a background, or “reset”, signal atthe node when no emission energy is present on the sensor (e.g., priorto application of excitation energy at the sample well) and to read asubsequent emission signal, for example.

According to some embodiments, correlated double sampling is employed toreduce background noise by subtracting a background or reset signallevel from the detected emission signal level. The collected emissionsignal and background signal associated with each segment of the sensormay be read out onto column lines 7-330. In some embodiments, anemission signal level and background signal are time-multiplexed onto acommon column line. There may be a separate column line for each sensorsegment. Signals from the column lines may be buffered and/or amplifiedwith amplification circuitry 7-340 (which may be located outside of anactive pixel array), and provided for further processing and analysis.In some embodiments the subtraction of the double-sampled signals iscalculated off-chip, e.g., by a system processor. In other embodiments,the subtraction may be performed on chip or in circuitry of theinstrument.

Some embodiments of correlated double sampling may operate by selectinga row to sample, wherein the sensors associated with the row haveintegrated signal charges over a sampling period and contain signallevels. The signal levels may be simultaneously read out onto thecolumns lines. After sampling the integrated signal levels, all thepixels in the selected row may be reset and immediately sampled. Thisreset level may be correlated to the next integrated signal that startsaccumulating after the reset is released, and finishes integrating aframe time later when the same row is selected again. In someembodiments, the reset values of the frame may be stored off-chip sothat when the signals have finished integrating and have been sampled,the stored correlated reset values can be subtracted.

In some embodiments, a sensor 2-122 with more than two segments mayrequire additional circuitry. FIG. 7-3C depicts signal-collection 7-312,amplification 7-320, and double-sampling circuitry associated with aquad sensor. According to some embodiments, signals from two or moresegments may be time-multiplexed onto a common signal channel at thepixel, as depicted in the drawing. The time-multiplexed signals mayinclude sampled background signals for each segment for noisecancellation. Additionally, the signals from two or more segments may betime-multiplexed onto a common column line.

According to some embodiments, temporal signal-acquisition techniquesmay be used to reduce background signal levels from an excitation sourceor sources, and/or discern different emissions from different emittersassociated with a sample. FIG. 7-4A depicts fluorescent emission anddecay from two different emitters that may be used to tag a sample,according to some embodiments. The two emissions have appreciablydifferent time-decay characteristics. A first time-decay curve 7-410from a first emitter may correspond to a common fluorescent moleculesuch as rhodamine. A second time-decay curve 7-420 may be characteristicof a second emitter, such as a quantum dot or a phosphorescent emitter.Both emitters exhibit an emission-decay tail that extends for some timeafter initial excitation of the emitter. In some embodiments,signal-collection techniques applied during the emission-decay tail maybe timed to reduce a background signal from an excitation source, insome embodiments, and to distinguish between the emitters, in someembodiments.

According to some implementations, time-delayed sampling may be employedduring the emission-decay tail to reduce a background signal due toradiation from an excitation source. FIG. 7-4B and FIG. 7-4C illustratetime-delay sampling, according to some embodiments. FIG. 7-4B depictsthe temporal evolution of an excitation pulse 7-440 of excitation energyfrom an excitation source, and a subsequent emission pulse 7-450 thatmay follow from a sample that is excited within the sample well. Theexcitation pulse 7-440 may result from driving the excitation sourcewith a drive signal 7-442 for a brief period of time, as depicted inFIG. 7-4C. For example, the drive signal may begin at a first time t₁and end at a second time t₂. The duration of the drive signal (t₂−t₁)may be between about 1 picosecond and about 50 nanoseconds, according tosome embodiments, though shorter durations may be used in someimplementations.

At a time t₃ following termination of the drive signal for theexcitation source, a sensor 2-260 (or sensor segment) at the pixel maybe gated to accumulate charge at a charge accumulation node 7-311 duringa second time interval extending from a time t₃ to a time t₄. The secondtime interval may be between about 1 nanosecond and about 50microseconds, according to some embodiments, though other durations maybe used in some implementations. As can be seen in reference to FIG.7-4B, a charge accumulation node will collect more signal charges due tothe emitting sample then due to the excitation source. Accordingly, animproved signal-to-noise ratio may be obtained.

Referring again to FIG. 7-4A, because of the different temporal emissioncharacteristics of the emitters, corresponding signals at a sensor maypeak at different times. In some implementations, signal-acquisitiontechniques applied during the emission-decay tail may be used to discerndifferent emitters. In some embodiments, temporal detection techniquesmay be used in combination with spatial and spectral techniques (asdescribed above in connection with FIG. 7-2, for example) to discerndifferent emitters.

FIG. 7-4D through FIG. 7-4H illustrate how double-sampling at a sensor,or sensor segment, can be used to distinguish between two emittershaving different temporal emission characteristics. FIG. 7-4D depictsemission curves 7-470, 7-475 associated with a first emitter and secondemitter, respectively. As an example, the first emitter may be a commonfluorophore such as rhodamine, and the second emitter may be a quantumdot or phosphorescent emitter.

FIG. 7-4E represents dynamic voltage levels at a charge accumulationnode 7-311 that may occur in response to the two different emissioncharacteristics of FIG. 7-4D. In the example, a first voltage curve7-472 corresponding to the fluorescent emitter may change more rapidly,because of the shorter emission span, and reach its maximum (or minimum,depending on the polarity of the node) at a first time t₁. The secondvoltage curve 7-477 may change more slowly due to the longer emissioncharacteristics of the second emitter, and reach its maximum (orminimum) at a second time t₂.

In some embodiments, sampling of the charge-accumulation node may bedone at two times t₃, t₄ after the sample excitation, as depicted inFIG. 7-4F. For example, a first read signal 7-481 may be applied to readout a first voltage value from the charge-accumulation node at a firsttime t₃. Subsequently, a second read signal 7-482 may be applied to readout a second voltage value from the charge-accumulation node at a secondtime t₄ without resetting the charge-accumulation node between the firstread and second read. An analysis of the two sampled signal values maythen be used to identify which of the two emitters provided the detectedsignal levels.

FIG. 7-4G depicts an example of two signals from the first read andsecond read that may be obtained for the first emitter having anemission curve 7-470 as depicted in FIG. 7-4D. FIG. 7-4H depicts anexample of two signals from the first read and second read that may beobtained for the second emitter having an emission curve 7-475 asdepicted in FIG. 7-4D. For example the sampling sequence shown in FIG.7-4F for the first emitter will sample the curve 7-472 and obtainapproximately the same values at the two read times. In the case of thesecond emitter, the sampling sequence depicted in FIG. 7-4F samples twodifferent values of the curve 7-477 at the two read times. The resultingpairs of signals from the two read times distinguish between the twoemitters, and can be analyzed to identify each emitter. According tosome embodiments, double sampling for background subtraction may also beexecuted to subtract a background signal from the first and second readsignals.

In operation, sensors 2-260 of a sensor chip may be subjected to awavelength calibration procedure prior to data collection from aspecimen to be analyzed. The wavelength calibration procedure mayinclude subjecting the sensors to different known energies havingcharacteristic wavelengths that may, or may not, correspond tofluorophore wavelengths that may be used with a sensor chip. Thedifferent energies may be applied in a sequence so calibration signalscan be recorded from the sensors for each energy. The calibrationsignals may then be stored as reference signals, that may be used toprocess real data acquisition and to determine what emission wavelengthor wavelengths are detected by the sensors.

V. Luminescent Markers

Embodiments may use any suitable luminescent markers to label samples(e.g., single molecules) in the specimen being analyzed. In someembodiments, commercially available fluorophores may be used. By way ofexample and not limitation, the following fluorophores may be used: AttoRho14 (“ATRho14”), Dylight 650 (“D650”), SetaTau 647 (“ST647”), CF 633(“C633”), CF 647 (“C647”), Alexa fluor 647 (“AF647”), BODIPY 630/650(“B630”), CF 640R (“C640R”) and/or Atto 647N (“AT647N”). Additionallyand/or optionally, luminescent markers may be modified in any suitableway to increase the speed and accuracy of the sample analysis process.For example, a photostabilizer may be conjugated to a luminescentmarker. Examples of photostabilizers include but not limited to oxygenscavengers or triplet-state quenchers. Conjugating photostabilizers tothe luminescent marker may increase the rate of photons emitted and mayalso reduce a “blinking” effect where the luminescent marker does notemit photons. In some embodiments, when a biological event occurs on themillisecond scale, an increased rate of photon emission may increase theprobability of detection of the biological event. Increased rates ofphoton events may subsequently increase the signal-to-noise ratio ofluminescence signal and increase the rate at which measurements aremade, leading to a faster and more accurate sample analysis.

VI. Excitation Sources

The excitation source 2-250 may be any suitable source that is arrangedto deliver excitation energy to at least one sample well 2-111 of theassay chip. Pixels on the assay chip may be passive source pixels. Theterm “passive source pixel” is used to refer to a pixel wherein theexcitation energy is delivered to the pixel from a region outside thepixel or pixel array of the assay chip, e.g., the excitation may be inthe instrument.

According to some embodiments, an excitation source may excite a samplevia a radiative process. For example, an excitation source may providevisible radiation (e.g., radiation having a wavelength between about 350nm and about 750 nm), near-infrared radiation (e.g., radiation having awavelength between about 0.75 micron and about 1.4 microns), and/orshort wavelength infrared radiation (e.g., radiation having a wavelengthbetween about 1.4 microns and about 3 microns) to at least oneexcitation region 3-215 of at least one sample well of the assay chip.In some embodiments, a radiative excitation source may provide energy toexcite an intermediary (e.g., a molecule, a quantum dot, or a layer ofmaterial comprising selected molecules and/or quantum dots) that isimmediately adjacent an excitation region of a sample well. Theintermediary may transfer its energy to a sample via a non-radiativeprocess (e.g., via FRET or DET).

In some embodiments, an excitation source may provide more than onesource of excitation energy. For example, a radiative excitation sourcemay deliver excitation energies having two or more distinct spectralcharacteristics. As an example, a multi-color LED may emit energiescentered at two or more wavelengths, and these energies may be deliveredto an excitation region of a sample well.

In overview and according to some embodiments, an instrument may includeat least one excitation source 2-250 to provide excitation energy to atleast one excitation region of at least one sample well of the assaychip or to at least one intermediary that converts or couples theexcitation energy to at least one sample within one or more excitationregions. As depicted in FIG. 2-3, radiation excitation energy 2-251 froman excitation source 2-250 may impinge on a region around a sample well2-211, for example. In some embodiments, there may be excitationcoupling structures 2-223 that aid in concentrating the incidentexcitation energy within an excitation region 2-215 of the sample well.

An excitation source may be characterized by one or more distinctspectral bands each having a characteristic wavelength. Forinstructional purposes only, an example of spectral emission from anexcitation source is depicted in spectral graph of FIG. 8-1A. Theexcitation energy may be substantially contained within a spectralexcitation band 8-110. A peak wavelength 8-120 of the spectralexcitation band may be used to characterize the excitation energy. Theexcitation energy may also be characterized by a spectral distribution,e.g., a full-width-half-maximum (FWHM) value as shown in the drawing. Anexcitation source producing energy as depicted in FIG. 8-1A, may becharacterized as delivering energy at a wavelength of approximately 540nm radiation and having a FWHM bandwidth of approximately 55 nm.

FIG. 4-1B depicts spectral characteristics of an excitation source (orexcitation sources) that can provide two excitation energy bands to oneor more sample wells. According to some embodiments, a first excitationband 8-112 is at approximately 532 nm, and a second excitation band8-114 is at approximately 638 nm, as illustrated in the drawing. In someembodiments, a first excitation band may be at approximately 638 nm, anda second excitation band may be at approximately 650 nm. In someembodiments, a first excitation band may be at approximately 680 nm, anda second excitation band may be at approximately 690 nm. According tosome embodiments, the peaks of the excitation bands may be within ±5 nmof these values.

In some cases, a radiative excitation source may produce a broadexcitation band as depicted in FIG. 8-1A. A broad excitation band 8-110may have a bandwidth greater than approximately 20 nm, according to someembodiments. A broad excitation band may be produced by a light emittingdiode (LED), for example. In some implementations, a radiativeexcitation source may produce a narrow excitation band, as depicted inFIG. 8-1B. A narrow excitation band may be produced by a laser diode,for example, or may be produced by spectrally filtering an output froman LED.

In some embodiments, the excitation source may be a light source. Anysuitable light source may be used. Some embodiments may use incoherentsources and other embodiments may use coherent light sources. By way ofexample and not limitation, incoherent light sources according to someembodiments may include different types of light emitting diodes (LEDs)such as organic LEDs (OLEDs), quantum dots (QLEDs), nanowire LEDs, and(in)organic semiconductor LEDs. By way of example and not limitation,coherent light sources according to some embodiments may includedifferent types of lasers such as organic lasers, quantum dot lasers,vertical cavity surface emitting lasers (VCSELs), edge emitting lasers,and distributed-feedback (DFB) laser diodes. Additionally oralternatively, slab-coupled optical waveguide laser (SCOWLs) or otherasymmetric single-mode waveguide structures may be used. Additionally oralternatively, a solid state laser such as Nd:YAG or Nd:Glass, pumped bylaser diodes or flashlamps, may be used. Additionally or alternatively,a laser-diode-pumped fiber laser may be used. In some embodiments, theoutput of a laser excitation source may be doubled in frequency to halfthe wavelength, in a nonlinear crystal, or a Periodically Poled LithiumNiobate (PPLN) or other similar periodically poled nonlinear crystal.This frequency doubling process may allow use of efficient lasers togenerate wavelengths more suitable for excitation. There may be morethan one type of excitation source for an array of pixels. In someembodiments, different types of excitation sources may be combined. Theexcitation source may be fabricated according to conventionaltechnologies that are used to fabricate a selected type of excitationsource.

The characteristic wavelength of a source of excitation energy may beselected based upon a choice of luminescent markers that are used in theassay analysis. In some implementations, the characteristic wavelengthof a source of excitation energy is selected for direct excitation(e.g., single photon excitation) of a chosen fluorophore. In someimplementations, the characteristic wavelength of a source of excitationenergy is selected for indirect excitation (e.g., multi-photonexcitation or harmonic conversion to a wavelength that will providedirect excitation). In some embodiments, excitation energy may begenerated by a light source that is configured to generate excitationenergy at a particular wavelength for application to a sample well. Insome embodiments, a characteristic wavelength of the excitation sourcemay be less than a characteristic wavelength of corresponding emissionfrom the sample. In some implementations, a characteristic wavelength ofthe excitation source may be greater than a characteristic wavelength ofemission from the sample, and excitation of the sample may occur throughmulti-photon absorption.

The excitation source may include a battery or any other power supply,which may be located somewhere other than the integrated bioanalysisdevice. For example, the excitation source may be located in aninstrument and the power may be coupled to the integrated bioanalysisdevice via conducting wires and connectors.

VII. Method of Use, Instrument Operation and User Interface

The instrument 2-120 may be controlled using software and/or hardware.For example, the instrument may be controlled using a processing device1-123, such as an ASIC, an FPGA and/or a general purpose processorexecuting software.

FIG. 9-1 illustrates a flowchart of operation of the instrument 2-120according to some embodiments. After a user has acquired a specimen toanalyze, the user begins a new analysis at act 9-101. This may be doneby providing an indication to the instrument 2-120 via the userinterface 2-125 by, e.g., pressing a button. At act 9-103, theinstrument 2-120 checks whether the assay chip 2-110 from a previouslyperformed analysis is still inserted in the instrument 2-120. If it isdetermined that an old chip is present, then the power to excitationsource may be turned off at act 9-105, the user is prompted at act 9-107to eject the previous chip using an indicator of the user interface2-125 and the instrument 2-120 waits for the old chip to be ejected atact 9-109.

When the previous chip is ejected by the user, or if the instrument2-120 determined at act 9-103 that the previous chip was alreadyremoved, the user is prompted to insert a new assay chip 2-110 for thenew analysis at act 9-111. The instrument 2-120 then waits for the newassay chip 2-110 to be inserted at act 9-113. When the user inserts thenew chip, the user is prompted at act 9-115 by an indicator of the userinterface 2-125to place the specimen to be analyzed onto the exposed topsurface of the assay chip 2-110 and also prompted to close the lid onthe instrument 2-120. The instrument 2-120 then waits for the lid to beclosed at act 9-117. When the lid is closed by the user, at act 9-119the excitation source may be driven to produce excitation energy forexciting the sample portions of the specimen present in the sample wellsof the assay chip 2-110. At act 9-121, the emission energy from thesamples is detected by the sensor 2-122 and data from the sensor 2-122is streamed to the processing device 2-123 for analysis. In someembodiments, the data may be streamed to external computing device2-130. At act 2-123, the instrument 2-120 checks whether the dataacquisition is complete. The data acquisition may be complete after aparticular length of time, a particular number of excitation pulses fromthe excitation source or one a particular target has been identified.When the data acquisition is completed, the data analysis is finished at9-125.

FIG. 9-2 illustrates an example self-calibration routine according tosome embodiments. The calibration routine may be executed at anysuitable time prior to the analysis of a specimen. For example, it maybe done once by the manufacturer for each instrument prior to shipmentto the end user. Alternatively, the end user may perform a calibrationat any suitable time. As discussed above, the instrument 2-120 iscapable of distinguishing between emission energy having differentwavelengths emitted from different samples. The instrument 2-120 and/orcomputing device 2-130 may be calibrated with calibration associatedwith each particular color of light associated with, for example, aluminescent tag used to tag molecules of a specimen being analyzed. Inthis way, the precise output signal associated with a particular colormay be determined.

To calibrate the device, a calibration specimen associated with a singleluminescent tag is provided to the instrument 2-120 one at a time. Theself-calibration begins at act 9-201 when a user places a specimencomprising luminescent tags that emit emission energy of a singlewavelength on an assay chip 2-110 and inserts the assay chip 2-110 intothe instrument 2-120. Using the user interface 2-125, the user instructsthe instrument 2-120 to begin the self-calibration. In response, at act9-203, the instrument 2-120 runs the calibration analysis byilluminating the assay chip 2-110 with excitation energy and measuringthe single wavelength emission energy from the calibration specimen. Theinstrument 2-120 may then, at act 9-205, save the detection patternmeasured on the array of sub-sensors of the sensor 2-122 for each pixelof the sensor array. The detection pattern for each luminescent tag maybe considered a detection signature associated with the luminescent tag.In this way, the signatures may be used as a training data set used toanalyze the data received from unknown samples analyzed in subsequentanalysis runs.

The above calibration routine may then be executed for every calibrationspecimen associated with a single luminescent tag. In this way, eachsensor 2-122 of the array of pixels is associated with calibration datathat may be used to determine the luminescent tag present in a samplewell during a subsequent analysis implemented at act 9-207 after thecompetition of the calibration routine.

FIG. 9-3 further illustrates how the calibration data may be acquiredand used to analyze the data according to some embodiments. At act 9-301calibration data is obtained from the sensors. This may be done usingthe aforementioned self-calibration routine. At act 9-303, atransformation matrix is generated based on the calibration data. Thetransformation matrix maps sensor data to the emission wavelength of asample and is a m x n matrix, where m is the number of luminescent tagswith different emission wavelengths and n is the number of sub-sensorsused to detect the emission energy per pixel. Thus, each column of thetransformation matrix represents the calibration values for the sensor.For example, if there are four sub-sensors per pixel and five differentluminescent tags, then the transformation matrix is a 4×5 matrix (i.e.,four rows and five columns) and each column is associated with adifferent luminescent tag, the values in the column corresponding to themeasured values obtained from the sub-sensors during theself-calibration routine. In some embodiments, each pixel may have itsown transformation matrix. In other embodiments, the calibration datafrom at least some of the pixels may be averaged and all the pixels maythen use the same transformation matrix based on the averaged data.

At act 9-305, the analysis data associated with a bioassay is obtainedfrom the sensors. This may be done in any of the ways described above.At act 9-307, the wavelength of the emission energy and/or the identityof the luminescent tag may be determined using the transformation matrixand the analysis data. This may be done in any suitable way. In someembodiments, the analysis data is multiplied by the pseudo-inverse ofthe transformation matrix, resulting in a m×1 vector. The luminescenttag associated with the vector component with the maximum value may thenbe identified as the luminescent tag present in the sample well.Embodiments are not limited to this technique. In some embodiments, toprevent possible pathologies that may arise when the inverse of a matrixwith small values is taken, a constrained optimization routine, such asa least square method or a maximum likelihood technique, may beperformed to determine the luminescent tag present in the sample well.

The foregoing method of using the calibration data to analyze data fromthe sensors may be implement by any suitable processor. For example,processing device 2-123 of the instrument 2-120 may perform theanalysis, or computing device 2-130 may perform the analysis.

IX. Computing Device

FIG. 10 illustrates an example of a suitable computing systemenvironment 1000 on which embodiments may be implemented. For example,computing device 2-130 of FIG. 2-1 may be implemented according to thecomputing system environment 1000. Additionally, the computing systemenvironment 1000 may act as a control system that is programmed tocontrol the instrument to perform an assay. For example, the controlsystem may control the excitation source to emit and direct lighttowards the sample wells of the assay chip; control the sensors to allowdetection of emission light from one or more samples in the samplewells; and analyze signals from the sensors to identify, e.g., byanalyzing the spatial distribution of the emission energy, the samplepresent in a sample well. The computing system environment 1000 is onlyone example of a suitable computing environment and is not intended tosuggest any limitation as to the scope of use or functionality of theinvention. Neither should the computing environment 1000 be interpretedas having any dependency or requirement relating to any one orcombination of components illustrated in the exemplary operatingenvironment 1000.

Embodiments are operational with numerous other general purpose orspecial purpose computing system environments or configurations.Examples of well-known computing systems, environments, and/orconfigurations that may be suitable for use with the invention include,but are not limited to, personal computers, server computers, hand-heldor laptop devices, multiprocessor systems, microprocessor-based systems,set top boxes, programmable consumer electronics, network PCs,minicomputers, mainframe computers, distributed computing environmentsthat include any of the above systems or devices, and the like.

The computing environment may execute computer-executable instructions,such as program modules. Generally, program modules include routines,programs, objects, components, data structures, etc. that performparticular tasks or implement particular abstract data types. Theinvention may also be practiced in distributed computing environmentswhere tasks are performed by remote processing devices that are linkedthrough a communications network. In a distributed computingenvironment, program modules may be located in both local and remotecomputer storage media including memory storage devices.

With reference to FIG. 10, an exemplary system for implementing theinvention includes a general purpose computing device in the form of acomputer 1010. Components of computer 1010 may include, but are notlimited to, a processing unit 1020, a system memory 1030, and a systembus 1021 that couples various system components including the systemmemory to the processing unit 1020. The system bus 1021 may be any ofseveral types of bus structures including a memory bus or memorycontroller, a peripheral bus, and a local bus using any of a variety ofbus architectures. By way of example, and not limitation, sucharchitectures include Industry Standard Architecture (ISA) bus, MicroChannel Architecture (MCA) bus, Enhanced ISA (EISA) bus, VideoElectronics Standards Association (VESA) local bus, and PeripheralComponent Interconnect (PCI) bus also known as Mezzanine bus.

Computer 1010 typically includes a variety of computer readable media.Computer readable media can be any available media that can be accessedby computer 1010 and includes both volatile and nonvolatile media,removable and non-removable media. By way of example, and notlimitation, computer readable media may comprise computer storage mediaand communication media. Computer storage media includes both volatileand nonvolatile, removable and non-removable media implemented in anymethod or technology for storage of information such as computerreadable instructions, data structures, program modules or other data.Computer storage media includes, but is not limited to, RAM, ROM,EEPROM, flash memory or other memory technology, CD-ROM, digitalversatile disks (DVD) or other optical disk storage, magnetic cassettes,magnetic tape, magnetic disk storage or other magnetic storage devices,or any other medium which can be used to store the desired informationand which can accessed by computer 1010. Communication media typicallyembodies computer readable instructions, data structures, programmodules or other data in a modulated data signal such as a carrier waveor other transport mechanism and includes any information deliverymedia. The term “modulated data signal” means a signal that has one ormore of its characteristics set or changed in such a manner as to encodeinformation in the signal. By way of example, and not limitation,communication media includes wired media such as a wired network ordirect-wired connection, and wireless media such as acoustic, RF,infrared and other wireless media. Combinations of the any of the aboveshould also be included within the scope of computer readable media.

The system memory 1030 includes computer storage media in the form ofvolatile and/or nonvolatile memory such as read only memory (ROM) 1031and random access memory (RAM) 1032. A basic input/output system 1033(BIOS), containing the basic routines that help to transfer informationbetween elements within computer 1010, such as during start-up, istypically stored in ROM 1031. RAM 1032 typically contains data and/orprogram modules that are immediately accessible to and/or presentlybeing operated on by processing unit 1020. By way of example, and notlimitation, FIG. 10 illustrates operating system 1034, applicationprograms 1035, other program modules 1036, and program data 1037.

The computer 1010 may also include other removable/non-removable,volatile/nonvolatile computer storage media. By way of example only,FIG. 10 illustrates a hard disk drive 1041 that reads from or writes tonon-removable, nonvolatile magnetic media, a magnetic disk drive 1051that reads from or writes to a removable, nonvolatile magnetic disk1052, and an optical disk drive 1055 that reads from or writes to aremovable, nonvolatile optical disk 1056 such as a CD ROM or otheroptical media. Other removable/non-removable, volatile/nonvolatilecomputer storage media that can be used in the exemplary operatingenvironment include, but are not limited to, magnetic tape cassettes,flash memory cards, digital versatile disks, digital video tape, solidstate RAM, solid state ROM, and the like. The hard disk drive 1041 istypically connected to the system bus 1021 through an non-removablememory interface such as interface 1040, and magnetic disk drive 1051and optical disk drive 1055 are typically connected to the system bus1021 by a removable memory interface, such as interface 1050.

The drives and their associated computer storage media discussed aboveand illustrated in FIG. 10, provide storage of computer readableinstructions, data structures, program modules and other data for thecomputer 1010. In FIG. 10, for example, hard disk drive 1041 isillustrated as storing operating system 1044, application programs 1045,other program modules 1046, and program data 1047. Note that thesecomponents can either be the same as or different from operating system1034, application programs 1035, other program modules 1036, and programdata 1037. Operating system 1044, application programs 1045, otherprogram modules 1046, and program data 1047 are given different numbershere to illustrate that, at a minimum, they are different copies. A usermay enter commands and information into the computer 1010 through inputdevices such as a keyboard 1062 and pointing device 1061, commonlyreferred to as a mouse, trackball or touch pad. Other input devices (notshown) may include a microphone, joystick, game pad, satellite dish,scanner, or the like. These and other input devices are often connectedto the processing unit 1020 through a user input interface 1060 that iscoupled to the system bus, but may be connected by other interface andbus structures, such as a parallel port, game port or a universal serialbus (USB). A monitor 1091 or other type of display device is alsoconnected to the system bus 1021 via an interface, such as a videointerface 1090. In addition to the monitor, computers may also includeother peripheral output devices such as speakers 1097 and printer 1096,which may be connected through a output peripheral interface 1095.

The computer 1010 may operate in a networked environment using logicalconnections to one or more remote computers, such as a remote computer1080. The remote computer 1080 may be a personal computer, a server, arouter, a network PC, a peer device or other common network node, andtypically includes many or all of the elements described above relativeto the computer 1010, although only a memory storage device 1081 hasbeen illustrated in FIG. 10. The logical connections depicted in FIG. 10include a local area network (LAN) 1071 and a wide area network (WAN)1073, but may also include other networks. Such networking environmentsare commonplace in offices, enterprise-wide computer networks, intranetsand the Internet.

When used in a LAN networking environment, the computer 1010 isconnected to the LAN 1071 through a network interface or adapter 1070.When used in a WAN networking environment, the computer 1010 typicallyincludes a modem 1072 or other means for establishing communicationsover the WAN 1073, such as the Internet. The modem 1072, which may beinternal or external, may be connected to the system bus 1021 via theuser input interface 1060, or other appropriate mechanism. In anetworked environment, program modules depicted relative to the computer1010, or portions thereof, may be stored in the remote memory storagedevice. By way of example, and not limitation, FIG. 10 illustratesremote application programs 1085 as residing on memory device 1081. Itwill be appreciated that the network connections shown are exemplary andother means of establishing a communications link between the computersmay be used.

VIII. Conclusion

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated that various alterations,modifications, and improvements will readily occur to those skilled inthe art.

Such alterations, modifications, and improvements are intended to bepart of this disclosure, and are intended to be within the spirit andscope of the invention. Further, though advantages of the presentinvention are indicated, it should be appreciated that not everyembodiment of the invention will include every described advantage. Someembodiments may not implement any features described as advantageousherein and in some instances. Accordingly, the foregoing description anddrawings are by way of example only.

The above-described embodiments of the present invention can beimplemented in any of numerous ways. For example, the embodiments may beimplemented using hardware, software or a combination thereof. Whenimplemented in software, the software code can be executed on anysuitable processor or collection of processors, whether provided in asingle computer or distributed among multiple computers. Such processorsmay be implemented as integrated circuits, with one or more processorsin an integrated circuit component, including commercially availableintegrated circuit components known in the art by names such as CPUchips, GPU chips, microprocessor, microcontroller, or co-processor.Alternatively, a processor may be implemented in custom circuitry, suchas an ASIC, or semicustom circuitry resulting from configuring aprogrammable logic device. As yet a further alternative, a processor maybe a portion of a larger circuit or semiconductor device, whethercommercially available, semi-custom or custom. As a specific example,some commercially available microprocessors have multiple cores suchthat one or a subset of those cores may constitute a processor. Though,a processor may be implemented using circuitry in any suitable format.

Further, it should be appreciated that a computer may be embodied in anyof a number of forms, such as a rack-mounted computer, a desktopcomputer, a laptop computer, or a tablet computer. Additionally, acomputer may be embedded in a device not generally regarded as acomputer but with suitable processing capabilities, including a PersonalDigital Assistant (PDA), a smart phone or any other suitable portable orfixed electronic device.

Also, a computer may have one or more input and output devices. Thesedevices can be used, among other things, to present a user interface.Examples of output devices that can be used to provide a user interfaceinclude printers or display screens for visual presentation of outputand speakers or other sound generating devices for audible presentationof output. Examples of input devices that can be used for a userinterface include keyboards, and pointing devices, such as mice, touchpads, and digitizing tablets. As another example, a computer may receiveinput information through speech recognition or in other audible format.

Such computers may be interconnected by one or more networks in anysuitable form, including as a local area network or a wide area network,such as an enterprise network or the Internet. Such networks may bebased on any suitable technology and may operate according to anysuitable protocol and may include wireless networks, wired networks orfiber optic networks.

Also, the various methods or processes outlined herein may be coded assoftware that is executable on one or more processors that employ anyone of a variety of operating systems or platforms. Additionally, suchsoftware may be written using any of a number of suitable programminglanguages and/or programming or scripting tools, and also may becompiled as executable machine language code or intermediate code thatis executed on a framework or virtual machine.

In this respect, the invention may be embodied as a computer readablestorage medium (or multiple computer readable media) (e.g., a computermemory, one or more floppy discs, compact discs (CD), optical discs,digital video disks (DVD), magnetic tapes, flash memories, circuitconfigurations in Field Programmable Gate Arrays or other semiconductordevices, or other tangible computer storage medium) encoded with one ormore programs that, when executed on one or more computers or otherprocessors, perform methods that implement the various embodiments ofthe invention discussed above. As is apparent from the foregoingexamples, a computer readable storage medium may retain information fora sufficient time to provide computer-executable instructions in anon-transitory form. Such a computer readable storage medium or mediacan be transportable, such that the program or programs stored thereoncan be loaded onto one or more different computers or other processorsto implement various aspects of the present invention as discussedabove. As used herein, the term “computer-readable storage medium”encompasses only a computer-readable medium that can be considered to bea manufacture (i.e., article of manufacture) or a machine. Alternativelyor additionally, the invention may be embodied as a computer readablemedium other than a computer-readable storage medium, such as apropagating signal.

The terms “program” or “software” are used herein in a generic sense torefer to any type of computer code or set of computer-executableinstructions that can be employed to program a computer or otherprocessor to implement various aspects of the present invention asdiscussed above. Additionally, it should be appreciated that accordingto one aspect of this embodiment, one or more computer programs thatwhen executed perform methods of the present invention need not resideon a single computer or processor, but may be distributed in a modularfashion amongst a number of different computers or processors toimplement various aspects of the present invention.

Computer-executable instructions may be in many forms, such as programmodules, executed by one or more computers or other devices. Generally,program modules include routines, programs, objects, components, datastructures, etc. that perform particular tasks or implement particularabstract data types. Typically the functionality of the program modulesmay be combined or distributed as desired in various embodiments.

Also, data structures may be stored in computer-readable media in anysuitable form. For simplicity of illustration, data structures may beshown to have fields that are related through location in the datastructure. Such relationships may likewise be achieved by assigningstorage for the fields with locations in a computer-readable medium thatconveys relationship between the fields. However, any suitable mechanismmay be used to establish a relationship between information in fields ofa data structure, including through the use of pointers, tags or othermechanisms that establish relationship between data elements.

Various aspects of the present invention may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

Also, the invention may be embodied as a method, of which an example hasbeen provided. The acts performed as part of the method may be orderedin any suitable way. Accordingly, embodiments may be constructed inwhich acts are performed in an order different than illustrated, whichmay include performing some acts simultaneously, even though shown assequential acts in illustrative embodiments.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing,” “involving,” andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

What is claimed is:
 1. A method of identifying a sample, the methodcomprising: delivering optical excitation energy to the sample locatedwithin a sample well in an array of sample wells arranged on a surfaceof a chip; obtaining, using at least one sensor, at least one signalindicative of luminescence emitted by the sample, wherein the chipcomprises a plurality of refractive lenses including a refractive lensconfigured to focus light emitted from the sample well towards the atleast one sensor; and using the at least one signal to identify at leasta portion of the sample.
 2. The method of claim 1, wherein deliveringoptical excitation energy further comprises delivering a plurality ofpulses of optical excitation energy to the sample well.
 3. The method ofclaim 1, wherein at least some refractive lenses in the plurality ofrefractive lenses each overlap with a respective sample well in thearray.
 4. The method of claim 3, wherein each of the at least somerefractive lenses is centered below its respective sample well.
 5. Themethod of claim 1, wherein individual refractive lenses of the pluralityof refractive lenses have a convex surface proximate to the array ofsample wells and a planar surface distal from the array of sample wells.6. The method of claim 1, wherein the plurality of refractive lenses isformed within a lens layer of the chip, and the chip further comprises alayer of dielectric material positioned between the lens layer and thearray of sample wells.
 7. The method of claim 1, wherein the sample is abiological sample identifiable by luminescence emitted from thebiological sample in response to illuminating the biological sample withthe optical excitation energy.
 8. The method of claim 7, wherein usingthe at least one signal to identify at least a portion of the samplefurther comprises identifying luminescently labeled subunits of thebiological sample.
 9. The method of claim 8, wherein the at least onesensor is configured to generate signals unique to different types ofthe luminescently labeled subunits.
 10. The method of claim 7, whereinthe biological sample comprises a target nucleic acid molecule, andusing the at least one signal to identify at least a portion of thesample further comprises identifying a sequence of the target nucleicacid molecule.
 11. The method of claim 10, wherein identifying thesequence of the target nucleic acid molecule comprises analyzing a timesequence of incorporation of luminescently labeled nucleotides and/ornucleotide analogs incorporated sequentially into a nucleic acidmolecule complementary to the target nucleic acid molecule.
 12. A methodfor nucleic acid sequencing comprising: delivering optical excitationenergy to a sample well in an array of sample wells arranged on asurface of a chip; exposing a complex having a template nucleic acidmolecule, a primer, and a polymerizing enzyme to luminescently labelednucleotides and/or nucleotide analogs, wherein the complex is located inthe sample well; obtaining, using at least one sensor, at least onesignal indicative of luminescence emitted by at least some of theluminescently labeled nucleotides and/or nucleotide analogs, wherein thechip further comprises a plurality of refractive lenses including arefractive lens configured to focus luminescence emitted from the samplewell towards the at least one sensor; and using the at least one signalto determine a series of incorporation events of nucleotides and/ornucleotide analogs into a nucleic acid molecule complementary to thetemplate nucleic acid molecule.
 13. The method of claim 12, whereindelivering optical excitation energy further comprises delivering aplurality of pulses of optical excitation energy to the sample well. 14.The method of claim 12, wherein at least some refractive lenses in theplurality of refractive lenses each overlap with a respective samplewell in the array.
 15. The method of claim 14, wherein each of the atleast some refractive lenses is centered below its respective samplewell.
 16. The method of claim 12, wherein individual refractive lensesof the plurality of refractive lenses have a convex surface proximate tothe array of sample wells and a planar surface distal from the array ofsample wells.
 17. The method of claim 12, further comprising identifyinga sequence of the target nucleic acid molecule by analyzing a timesequence of incorporation of nucleotides and/or nucleotide analogsincorporated sequentially into the nucleic acid molecule complementaryto the target nucleic acid molecule.
 18. The method of claim 12, whereinthe at least one sensor is configured to generate signals unique todifferent types of the luminescently labeled nucleotides and/ornucleotide analogs.
 19. The method of claim 12, wherein the polymerizingenzyme is immobilized in the sample well.
 20. The method of claim 12,wherein obtaining the at least one signal further comprises reading outthe at least one signal from the at least one sensor.